Targeting Tumor Markers with Antisense Peptides: An Example of Human Prostate Specific Antigen.

The purpose of this paper was to outline the development of short peptide targeting of the human prostate specific antigen (hPSA), and to evaluate its effectiveness in staining PSA in human prostate cancer tissue. The targeting of the hPSA antigen by means of antisense peptide AVRDKVG was designed according to a three-step method involving: 1. The selection of the molecular target (hPSA epitope), 2. the modeling of an antisense peptide (paratope) based on the epitope sequence, and 3. the spectroscopic evaluation of sense-antisense peptide binding. We then modified standard hPSA immunohistochemical staining practice by using a biotinylated antisense peptide instead of the standard monoclonal antibody and compared the results of both procedures. Immunochemical testing on human tissue showed the applicability of the antisense peptide technology to human molecular targets. This methodology represents a new approach to deriving peptide ligands and potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

Correlation of the HPV detection, protein expression and DNA content in cutaneous pre-invasive and invasive carcinoma among Croatian patients.

Development of cutaneous carcinomas has been associated with HPV infection. There have been various reports on p16, p53 and pRb expression in cutaneous carcinomas and on its linkage to HPV status. Association of protein expression and HPV infection with DNA content is not clear. The aim of this study was to determine a possible correlation between HPV type, protein expression and DNA content in both pre-invasive and invasive squamous cell carcinoma, as well as differences between studied groups in these parameters. Sections of formalin fixed paraffin-embedded tumor tissue from 54 cases of Morbus Bowen (preinvasive cutaneous carcinoma) and 41 cases of invasive squamous cell carcinoma of the skin were subjected to HPV genotyping using Lipa (Line imuno probe assay), immunohistochemical staining for p16(INK4A), p53, pRb and prepared for flow cytometry DNA content analysis. Obtained data were analyzed in SPSS using Chi square test. Only p16 expression showed statistically significant differences in studied groups. Statistically significant correlations were found only in MB between parameters HPV-p53, p53-pRb and p53-p16. Our results suggest different virus-induced pathobiology pathways for different cutaneous carcinoma groups.

Effect of chorioamnionitis on mortality, early onset neonatal sepsis and bronchopulmonary dysplasia in preterm neonates with birth weight of < 1,500 grams.

The aim of the study was to investigate the effects of chorioamnionitis on mortality and early onset neonatal sepsis (EONS) and bronchopulmonary dysplasia (BPD) in preterm neonates with birth weight < or = 1,500 g. The study included 395 preterm infants born at the Zagreb Clinical Hospital Center, from January 2001 to December 2005. All the placentas from preterm deliveries were sent for pathological examination. The patients were categorized into two groups: one including patients with chorioamnionitis at placental histology (47%) and the other control group without chorioamnionitis (53%). Neonates were distributed into 3 groups according to gestational age: the first group with 132 (33%) infants born at < or = 28 weeks of gestation, the second with 202 (52%) infant born from 29 to 32 weeks of gestation and the third with 61 (15%) infants born at > or = 33 weeks gestation. Chorioamnionitis was diagnosed significantly more often in the first gestational age group (91/132-69% of infants, chi2 = 51.307, p < 0.05). The outcome was lethal in 67/395 (17%) patients; 55% of them had chorioamnionitis (chi2 = 2.421, p > 0.05). Lethal outcome ensued in 54/132 (41%) infants from the first gestational age group; 30/54 (55%) were born from pregnancies complicated by chorioamnionitis. In comparison with the control group, mortality was significantly higher in the group of premature infants with gestation < or = 28 weeks whose placentas showed chorioamnionitis (chi2 = 7.645, p < 0.01). EONS was probable or confirmed in 100/395 (25%) infants; in 66/100 (66%) infants pregnancy was complicated by chorioamnionitis (chi2 = 22.396, p < 0.01). BPD developed in 25/395 (6%) infants; in 12/25 (48%) infants placentas showed chorioamnionitis (chi2 = 0.022, p > 0.05). In conclusion, premature neonates from pregnancies complicated by chorioamnionitis are more often born at < or = 28 weeks of gestation. Chorioamnionitis in neonates whose gestation is < or = 28 weeks leads to a significantly higher rate of mortality than in neonates with a longer gestation period. A greater incidence of EONS was proven in the group of infants with chorioamnionitis. The difference between the incidence of BPD in preterm infants born from pregnancies complicated by chorioamnionitis and the control group was not significant.

[Clinical recommendations for diagnosing, treatment and monitoring of patients with endometrial cancer -- Croatian Oncology Society and Croatian Society for Gynecology and Obstetrics as Croatian Medical Association units and Croatian Society of Gynecological Oncology]. / Klinicke upute za dijagnostiku, lijecienje i pracenje bolesnica oboljelih od raka maternice hrvatskoga onkoloskog drustva i hrvatskog drustva za ginekologiju i opstetriciju hrvatskoga lijecni(ckog zbora te hrvatskoga ginekoloskoonkoloskog drustva.

Uterine cancer occurs mainly in postmenopausal women, usually as vaginal bleeding. Following ovarian and cervical cancer it is the third most common cause of female reproductive system cancer death. Diagnosis is set by analyzing samples obtained via hysterectomy with salpingo-oophorectomy and pelvic / paraaortal lymphadenectomy. The following text presents the clinical guidelines in order to standardize the procedures and criteria for the diagnosis, treatment and monitoring of patients with uterine cancer in the Republic of Croatia.

[Clinical recommendations for diagnosing, treatment and monitoring of patients with ovarian cancer -- Croatian Oncology Society and Croatian Society for Gynecology and Obstetrics as Croatian Medical Association units and Croatian Society of Gynecological Oncology]. / Klinicke upute za dijagnostiku, lijecenje i pracenje bolesnica oboljelih od raka jajnika hrvatskoga onkoloskog drustva i hrvatskog drustva za ginekologiju i opstetriciju hrvatskoga lijetnickog zbora te hrvatskoga ginekoloskoonkoloskog drustva.

Ovarian cancer together with fallopian tube represents the fifth most common female cancer in the Republic of Croatia. Epithelial ovarian cancer, serous subtype, encompasses most of malignant ovarian neoplasms. Less common are various non-epithelial ovarian malignancies. A special group consists of epithelial carcinomas of low malignant potential with clinically indolent flow, good prognosis and no invasion, and primary cancer of the peritoneum and fallopian tube cancer. Clinically, these malignant tumors are generally asymptomatic in early stages, and usually diagnosed in advanced stages. The diagnosis is confirmed by pathological examination, and occasionally, cytological findings after completing diagnostic procedures. Multidisciplinary team makes treatment decisions, taking into account age, general condition and comorbidities of the patient and characteristics of the tumor itself, including disease stage, histological type and grade of the tumor. The principles of treatment of primary peritoneal and fallopian tube cancer are based on the principles of treatment of epithelial ovarian cancer involving surgery, chemotherapy, immune and hormone therapy, and symptomatic-supportive care throughout the treatment. Less common histological types have a different treatment approach being more frequently diagnosed in the early stages of the disease, have more indolent flow, so in these patients conservative surgeries with the goal of preserving fertility are more often employed. The following text presents the clinical guidelines in order to standardize the procedures and criteria for the diagnosis, management, treatment and monitoring of patients with ovarian carcinoma, fallopian tube and primary peritoneal cancer in the Republic of Croatia.

[Clinical recommendations for diagnosing, treatment and monitoring of patients with uterine cervical cancer -- Croatian Oncology Society and Croatian Society for Gynecology and Obstetrics as Croatian Medical Association units and Croatian Society of Gynecological Oncology]. / Klinicke upute za dijagnostiku, lijecenje i pracenje bolesnica oboljelih od raka vrata maternice hrvatskoga onkoloskog drustva i hrvatskog drustva za ginekologiju i opstetriciju hrvatskoga lije nickog zbora te hrvatskoga ginekoloskoonkoloskog drustva.

Cervical cancer, in comparison with other gynecological malignancies, mainly affects younger women. It can be prevented trough educational programs, screening and early detection. It also can be efficiently treated when it appears. Treatment modalities include surgery, chemotherapy and radiotherapy, according to the stage of the disease and patient condition. Treatment decisions should be made after multidisciplinary team discussion. Due to the significance of this disease it is important to define and implement standardized approach for diagnostic, treatment and monitoring algorithm as well. The following text presents the clinical guidelines in order to standardize the procedures and criteria for the diagnosis, management, treatment and monitoring of patients with uterine cervical cancer in the Republic of Croatia.

A low-grade ovarian carcinoma case with coincident LOH of PTCH1 and BRCA1, and a mutation in BRCA1.

We report a case of a 53-year-old woman with Grade 1 serous cystadenocarcinoma on her left ovary and metastatic serous adenocarcinoma on her right ovary. Serous carcinoma is the most common type of ovarian cancer, representing approximately half of all cases. Because of positive family history, the patient was referred for BRCA1/2 screening. Germline BRCA1 mutation c.676delT (p.C226VfsX8) was found, and in tumor tissue the normal allele was lost. Tumor tissue also had loss of heterozygosity in the PTCH1 gene, one of the major members of the Hedgehog-Gli (Hh-Gli) pathway. Gene expression analysis showed upregulation of the Hh-Gli pathway in both ovaries compared with healthy ovarian tissue. Primary cell culture was developed from the patient's tissue and showed downregulation of gene expression in response to cyclopamine, a Hh-Gli pathway inhibitor. The Hh-Gli signaling pathway may play a role in malignant transformation and metastasis of ovarian cancer.

Expression of Ki-67, P53 and progesterone receptors in uterine smooth muscle tumors. Diagnostic value.

Aim was to investigate expression of Ki-67, P53 and progesterone receptors (PR) in leiomyomas (LM), smooth muscle tumors of uncertain malignant potential (STUMP) and leiomyosarcomas (LMS) and to establish possible usefulness of these three parameters in distinguishing between LM and STUMP and STUMP and LMS. Retrospective study of 51 uterine smooth muscle neoplasm (16 LM, 18 STUMP, 17 LMS) technically acceptable for analyses from years 2002-2007 from Department of Gynecological and Prenatal Pathology, University Hospital Center Zagreb, Croatia. Immunohistochemical analysis of Ki-67, P53 and PR expression was performed. Every nuclei stained brown, regardless of shade intensivity, was considered positive. The interpretation of immunohistochemical staining was expressed as number of positive cells in 100 cell count in most active area of the slide. Non-parametric analysis of variance Kruskal-Walis test was performed. Ki-67 expression was negative in all LM and higher than 5% in 12/18 STUMP and 10/17 LMS. Significant differences were observed between LM and STUMP expression for Ki-67 (p = 0.000), and LM and LMS expression for Ki-67 (p = 0.000). There was no expression of P53 in LM, expression of P53 was found in 7/17 LMS and 5/18 STUMP Expression of P53 was significant between LM and LMS (p = 0.002), and between LM and STUMP (p = 0.006). Expression of PR was found in 16/16 LM and 18/18 STUMP 10/17 LMS did not show PR expression. Expression of PR was significant between LM and LMS (p = 0.018) and STUMP and LMS (p = 0.004). The findings of our study in concordance with other study results are helpful information establishing more diagnostic criteria and parameters for diagnosis in doubtful cases between three entities. Immunoassaying for Ki-67, P53 and PR are such parameters. The panel of their expression in specific case eases diagnosis.

Vascular endothelial growth factor and intratumoral microvessel density as prognostic factors in endometrial cancer.

The aim of this research was to determine the VEGF A expression in tumor cells and the intratumoral microvessel density and their prognostic significance in the survival of the subjects. 87 subjects were monitored retrospectively for a period of 60 to 132 months. The subjects were treated at the Department of Obstetrics and Gynecology of Osijek University Hospital Center, Croatia. We analysed standard clinical, pathohistological and therapeutical prognostic factors, intratumoral microvessel density and expression of VEGF A. Five-year survival was calculated by the life chart method and presented graphically by Kaplan-Meier curves. Reaching conclusions on statistical hypotheses in this paper was done with a reliability level p < 0.05. Of the analyzed clinical prognostic factors, those which proved to be statistically significant and independent prognostic factors were age and clinical stage of the disease, and of pathohistologic ones it was the depth of myometrial invasion and VEGF expression. An elevated VEGF expression is associated with deep myometrial invasion, poorly differentiated tumors, histologic type and intratumoral microvessel density to a statistically significant degree. Elevated VEGF expression, age, FIGO stage and depth of myometrial invasion play a significant prognostic role in patients with endometrial cancer. VEGF receptors could be a target for adjuvant therapy in VEGF positive endometrial cancer.

Cervix cancer screening in Croatia within the European Cervical Cancer Prevention Week.

Croatia still has opportunistic screening and the organized national screening has been planned. The European Cervical Cancer Prevention Week was held twice in Croatia, in January 2008 and 2009. Within the first one in 2008, information campaign "For All Women" via mass media was held, and women were invited to the organized free gynecological examination and Papanicolaou test (Pap test) in the University Department of Gynecology and Obstetrics, Zagreb University Hospital Center. Following invitation 481 women attended the testing; the median age was 55 years. There were more women aged > or = 50 (n = 353), with the highest participation in the age group 55-59 years (n = 94). Some women came because of subjective symptoms (n = 10), but the majority of them came only for testing (n = 471). According to history of previous cytological testing, 400 women have had > or = 1 negative findings, 71 women have had > or = 1 positive findings, 9 women attended Pap test for the first time, and 1 woman does not know about previous testing. Cervical cytology was abnormal in 35 women (7.28%), the median age was 42 years with the highest proportion in the age group 30-34 years (n = 7); among all of them 21 women (60%) had no abnormal Pap test previously. The findings were: Atypical squamous cells of undetermined significance--ASC-US (n = 9), ASC cannot exclude high-grade squamous intraepithelial lesion--ASC-H (n = 1), cervical intraepithelial neoplasia--CIN 1 (n = 13), CIN 2 (n = 1), CIN 3 (n = 6), carcinoma planocellulare (n = 2), atypical glandular cells--AGC-favor reactive endocervical cells (n = 3). Among women aged < or = 49 there were 20.47% abnormal findings and among those aged > or = 50, 2.55%. According to 21 positive Pap tests previously, among women aged < or = 49 there were 30.71% while among those aged > or = 50 there were 9.07%. Within the European Cervical Cancer Prevention Week in 2009, employed women from one national company were invited by internal information to the same procedure. A smaller group of younger asymptomatic women came for testing (n = 53), median age 39 years. According to history of previous cytological testing, 50 women have had > or = 1 negative findings, 3 women have had > or = 1 positive findings. In this study, Pap test was positive in 3.77% (n = 2). National screening programme should be focused on the participation of all personally invited women, especially younger age groups and under-screened women. Well designed information campaign should be implemented in national screening programme.

Cytologic characteristics of adenoid cystic carcinoma of the cervix uteri--case report.

Adenoid cystic carcinoma is a rare malignancy, usually found in the salivary glands, although this unusual tumor can be found at other sites of the body. In particular, regarding adenoid cystic carcinoma of the cervix (ACCC) most frequently reports are given for postmenopausal women. In this respect, our work is one among very few in the literature that considers a cytologic picture of this uncommon cervix carcinoma. The case of 74 year old patient with postmenopausal bleeding is described. Both Pap smear and air dried smear of the uterine cervix were analyzed. The cytologic findings revealed very few small clusters of abnormal glandular cells, as well as some amorphous eosinophilic globule-like material, with granulomatous and necrotic background. The latter includes a lot of histiocytes, multinucleated giant cells, large aggregates of epitheloid cells and lymphocytes. Histology revealed the diagnosis of ACCC. We emphasize the importance of careful screening of Pap smear that might be crucial in the case of suspicious clinical findings in postmenopausal women, when the possibility of ACCC has to be considered.

Cytology of cervical intraepithelial glandular lesions.

Cytological criteria for the identification of glandular intraepithelial lesions (GIL) have not yet been fully described, especially for the precursors of adenocarcinoma in situ (AIS), thus these lesions may frequently remain unrecognized. As most patients diagnosed with AIS or mild to moderate GIL (grades I, II) are free from clinical symptoms, cytology has a very responsible role in the detection of these lesions. The aim of the study was to achieve the most appropriate cytologic diagnosis of intraepithelial lesions of endocervical columnar epithelium, analyzing the cytology findings in patients with histologically verified AIS and GIL (I, II). The value of cytology in the detection and differential diagnosis was assessed in 123 patients with definitive histologic diagnosis of glandular lesions (AIS, n = 13; GIL I, n = 11; and GIL II, n = 7), and glandular lesions associated with squamous component (AIS associated with cervical intraepithelial neoplasia (CIN) or invasive squamous cell carcinoma (SCC), n = 58; GIL I or GIL II associated with CIN, n = 28; and GIL associated with microinvasive squamous carcinoma (MIC), n = 6). In 95.1% of patients, lesions were detected by cytologic analysis that indicated additional diagnostic procedure. In terms of differential diagnosis, cytology showed higher accuracy in predicting lesion severity vs. type of epithelial alteration (75.6% vs. 55.3%) and abnormalities of columnar epithelium (95.7%; vs. 74.2%). The accuracy of cytology was higher in pure (AIS, 61.5% and GIL I, II, 22.2%) than in mixed lesions (25.9% and 20.6%). Continuous improvement in cervical specimens and cytodiagnostic skills, better understanding of intraepithelial adenocarcinoma and precursors, and their inclusion in the classification of cytologic and histologic findings are expected to upgrade the detection of these lesions, and to reduce the invasive cervical adenocarcinoma morbidity and mortality.

Digital morphometry of cytologic aspirate endometrial samples.

Unlike cervical cytology, morphological cytology criteria in the differential diagnosis of endometrium have not yet been clearly defined, and methods to allow for more precise evaluation of endometrium status have been searched for. The aim of the present study was to assess the value of morphometric nucleus analysis of cytologic aspirate endometrial samples in proliferative, hyperplastic and malignant endometrium by use of digital image analysis. Morphometric analysis was performed on archival cytologic aspirate endometrial samples (at least 10 per group) stained according to Papanicolaou (n = 77) and May-Grünwald-Giemsa (MGG; n = 80) with the following histopathologic diagnoses: proliferative endometrium, hyperplasia simplex, hyperplasia complex, hyperplasia complex atypica, and adenocarcinoma endometriodes endometrii (grade I, II and III). Interactive image analysis (nuclear area, convex area, perimeter, maximum and minimum radius, length and breadth, as well as nucleus form factor and elongation factor) was performed by use of the SFORM software (VAMSTEC, Zagreb) on at least 50 (Papanicolaou stain) and 100 (MGG stain) well preserved endometrial epithelial cell nuclei without overlapping, at magnification of x1000. Statistical data analysis was done by use of the Statistica Ver. 6 statistical package. Multivariate analysis (ANOVA) distinguished malignant, hyperplastic and proliferative endometrium according to all morphometric variables with both staining methods (p < 0.05). However, on interactive testing of the groups (Kruskal-Wallis test), hyperplasias without atypia yielded no significant differences (p > 0.05) from atypical hyperplasia, adenocarcinoma and proliferative endometrium only according to the nucleus form factor and elongation factor (Papanicolaou stain), whereas malignant and atypical hyperplastic endometrium (MGG stain) differed statistically significantly (p < 0.05) from proliferative and hyperplastic endometrium without atypia according to all study parameters except for the nucleus form factor (p > 0.05). According to the cytologic staining method, morphometric parameters were considerably higher in MGG stained endometrial samples, reaching the level of statistical significance (p < 0.05) except for the nucleus form factor and elongation factor (p > 0.05) in the groups of hyperplasia simplex and complex, well differentiated adenocarcinoma (form factor) and atypical hyperplasia (elongation factor). A combination of cytomorphology and the morphometric variables assessed in this study can yield useful information on the cytologic state of endometrium, with special reference to the possible differentiation of the group of hyperplasia without atypia from the group of adenocarcinoma and atypical hyperplasia.

Liquid-based cytology--new possibilities in the diagnosis of cervical lesions.

Liquid-based cytology (LBC) enables the use of supplementary methods in the diagnosis and prognosis of cervical lesions. The aim of this study was to analyze the correlation between p16-INK4a immunoexpression in ThinPrep cervical cytologic samples and human papillomavirus (HPV) detection by polymerase chain reaction (PCR) from the same sample. LBC-ThinPrep (Cytyc, USA) cervical cytology samples, prepared and stained by Papanicolaou method, were analyzed using modified Bethesda cytologic classification named "Zagreb 2002". A second ThinPrep slide, prepared from the same sample, was immunostained for p16INK4a using CINtec p16INK4a Cytology Kit (DakoCytomation, Denmark). Increased expression of the high-risk (HR) HPV E6 and E7 oncogenes results in a highly specific increase in p16 protein expression and overexpression of p16INK4a acts as a potential biomarker for cervical cancer progression from premalignant lesions. Brown nuclear and/or cytoplasmic staining of abnormal cells was considered a positive result. Residual material was used for 13 HR HPV-DNA detection by the PCR based AMPLICOR HPV test (Roche Molecular Systems). A total of 120 ThinPrep Pap tests with the following cytologic diagnoses: 17 within normal limits, 17 atypical squamous cell (ASC) (7 ASC of undetermined significance /ASCUS/ and 10 ASC of high-grade squamous intraepithelial lesions cannot be excluded /ASC-H/), 26 low-grade squamous intraepithelial lesions (LSIL) corresponding cervical intraepithelial neoplasia (CIN) 1, 57 high-grade SIL (HSIL) i.e. 24 CIN II and 33 CIN III and 3 squamous cell carcinoma (SCC) were included in the study. All CIN III (n = 33) and SCC (n = 3) specimens expressed p16INK4a immunoreactivity, whereas the HR HPV test was positive in 97% (32/33) of CIN III and 100% (3/3) of SCC specimens. The p16INK4a biomarker was positive in 87.5% (21/24) of CIN II and 69% (18/26) of CIN I, while the HR HPV was positive in 75% (18/24) of CIN II and 50% (13/26) of CIN I. In ASCUS cytology, p16INK4a and HR HPV showed the same rate of positivity (28.5%; 2/7). Expression of p16INK4a was detected in all cytologic (10/10) ASC-H lesions, in contrast to HR HPV detected in only 20% (2/10) of ASC-H cases. These data suggest the p16INK4a evaluation in ThinPrep cervical samples to be significantly associated with HR HPV testing by PCR in the same sample for the diagnosis of HSIL lesions and cervical carcinomas. A prospective study with longer follow up may clarify the predictive values in the management of LSIL and ASC diagnosis.

Desmoplastic Small Round Cell Tumor of the Ovary: A Case Report with a New Modality of Treatment and Review of the Literature.

OBJECTIVE: Desmoplastic small round cell tumor (DSRCT) is a rare intraabdominal neoplasm that grows along serosal surfaces and is primarily found in young men. To date, only 16 cases of ovarian DSRCT have been previously reported in women in the English literature, and no large population-based studies on this topic exist. CASE REPORT: We report the case of a 19-year-old virgo with unremarkable past medical history, initially presented with abdominal fullness. After being treated with the optimal treatment modality (primary and secondary surgical debulking, unique chemotherapy, protocol and adjuvant radiotherapy), the patient has remained without tumor disease for 40 months. CONCLUSION: Although the best therapy for patients with DSRCT has yet to be determined, combining complete surgical resection, adjuvant chemotherapy, and radiotherapy is required to prolong survival and to achieve proper quality of life.

[Diagnostic-therapeutic guidelines for men whose partners have HPV genital infection]. / Dijagnosticko-terapijski postupnik za muskarce kojih partnerice imaju HPV-genitalnu infekciju.

Human papilloma virus infection is the most frequent sexually transmitted disease. HPV infections are connected with different diseases such as benign warts, condylomata acuminata, malignant cervical, vulvar, vaginal, penile and anal carcinoma. Peniscopy with HPV detection is a specific diagnostic method for diagnosis of subclinical HPV genital infection in asymptomatic men. Taking the samples for HPV detection from asymptomatic men with curette is more qualitative way of getting enough samples then taking swab with wooden stick or (tongue) depressor. Early diagnosis and treatment of HPV infections in men is of potential benefit because their eradication can reduce the viral reservoir and as the result of that the incidence of CIN, carcinoma in situ and invasive cervical carcinoma can be reduced. For the correct diagnosis and for choosing the adequate therapeutical technique, we suggest diagnostic-therapeutic guidelines for HPV genital infection in men.

[Proposal of cervical cancer early detection programme in Croatia]. / Prijedlog programa ranog otkrivanja raka vrata maternice u hrvatskoj.

Opportunistic screening by Pap smear was introduced in Croatia in 1950s, with a consequent decrease of cervical cancer incidence and mortality. Since 1990s, no further decrease has been observed, and there are still about 370 new cases and 100 deaths of cervical cancer yearly in Croatia. In scope of the proposed early detection programme, all Croatian women aged 25-64 years should be screened. In the first phase of the programme, the target population would be tested by Pap-smear every third year. In the second phase, HPV-testing would also be introduced for women over 30 years. Organization of the programme at county level is proposed, while the evaluation and monitoring would be performed both at county level and centrally. Regarding the present costs of treatment and sick-leave of cervical cancer patients, it is estimated that the introduction of cervical cancer screening programme in Croatia would be cost-effective already after the first decade.

[The value of searching for additional prognostic factors in combination with Nottingham Prognostic Index in breast carcinoma patients]. / Vrijednost istrazivanja dodatnih prognostickih cimbenika u kombinaciji s Nottinghamskim Prognostickim Indeksom u bolesnica s karcinomom dojke.

One of prognostic factors known as Nottingham Prognostic Index (NPI), which is combination of known prognostic factors such as tumor size, grade and axillary node status, is recently in usage in some European countries in clinical practice in prediction of breast carcinoma patients' survival. Therefore, the aim of this study was to verify, according to our experience, the prognostic significance of Nottingham Prognostic Index (NPI) in breast carcinoma patients in association with other new prognostic factors. In this study 148 consecutive specimens of breast carcinoma patients were analyzed. The following data for each patient were collected: age, tumor size, histological grade, axillary lymph node status, overall survival, estrogen (ER), progesterone (PR) receptor expression as well as expression of bcl-2, Ki-67, nm23, HER-2/neu, and p53. The Nottingham Prognostic Index (NPI) was calculated from the pathological information and patients were grouped according to the standard NPI index into: good prognostic group (GPG), moderate prognostic group (MPG), and poor prognostic group (PPG). The correlation of prognostic groups according to the NPI with other prognostic and predictive factors such as age, ER, PR, p53, bcl-2, Ki-67, nm23, Ki-67, Cathepsin D and HER-2/neu on overall survival was analyzed. The results of univariate analysis showed statistically significant correlation between patients' age, NPI prognostic groups and stage of disease with patients survival. When other prognostic factors were correlated with NPI prognostic groups there was not additional prognostic discrimination in given prognostic groups. Only marginally statistically significant influence of p53 expression was found on patient survival between MPG and PPG. It seems that other prognostic factors in combination with NPI prognostic groups do not have in our group of patients practical clinical relevance for the management of patients with breast carcinoma.

Comparison of polymerase chain reaction and catalyzed signal amplification in situ hybridization methods for human papillomavirus detection in paraffin-embedded cervical preneoplastic and neoplastic lesions.

BACKGROUND: Two molecular methods for HPV genotyping in formalin-fixed, paraffin-embedded tissue were evaluated: in house polymerase chain reaction assay (PCR) with consensus and type-specific primers and a novel procedure of in situ hybridization-a catalyzed signal amplification system (CSA-ISH, Genpoint, DAKO, Glostrup, Denmark). The number of HPV positive cases and detected viral types were compared in cervical biopsies and cone specimens according to histopathological diagnosis. Primer efficiency in detecting various types of HPV by PCR method was evaluated. METHODS: DNA samples (101) were used as a template to amplify with three pairs of consensus (MY09/11, GP5+/6 +, CPI/IIG) and four type-specific HPV primers (HPV-6/11, 18, 16 and 33). The according histological tissue sections were analyzed with CSA-ISH method, using commercial HPV biotinylated probes HPV-6/11, 16/18 and 31/33/51. RESULTS: The degree of concordance for PCR and CSA-ISH was 64.4%. In 63 of 101 samples (62.4%), HPV was detected by PCR, while only 35 (34.7%) were positive using CSA-ISH. CSA-ISH found lower percentages for all HPV types, except HPV-6/11. A lower percentage of positive results in all high-grade lesions was detected by CSA-ISH. Multiple infections were detected by PCR in only one sample and in three samples by CSA-ISH. Detection with My09/11 primers followed by Gp5+/6+ primers, in nested reaction, gave the highest number of positive results: 58 of 63 (92%). None of the samples diagnosed as condylomata planum or CIN I was positive for HPV-6/11 (low risk type), which was detected exclusively in condylomata acuminatum group. CONCLUSIONS: A significantly higher number of positive samples was detected with PCR than with CSA-ISH method. CSA-ISH method should be improved, especially in detecting HPV in high-grade lesions. CSA-ISH may be more accurate in detection of multiple infections. GP5+/6+ in nested reaction after MY09/11 detected the highest number of positive results. Samples diagnosed as benign lesions positive on HPV-X must be monitored as possible candidates for progression. CIN I lesions, which were HPV negative, probably will not progress. This finding may be important in planning therapy and avoiding unnecessary treatment.

Extraskeletal myxoid chondrosarcoma of the vulva with PLAG1 gene activation: molecular genetic characterization of 2 cases.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare mesenchymal neoplasm, rarely reported in the genitourinary tract with only 5 cases reported in the vulva. We investigated 2 cases of vulvar sarcomas whose morphologic appearance and immunohistochemical profiles were consistent with EMC using fluorescence in situ hybridization (FISH), reverse-transcription polymerase chain reaction, and a whole genome expression array. FISH and reverse-transcription polymerase chain reaction assays showed no EWSR1 and NR4A3 loci rearrangements. Microarray-based analysis also revealed no changes in NR4A3 and EWSR1 gene transcription levels. Microarray data showed a significant downregulation of the muscle-related genes (eg, myosin heavy chain family, actins, myoglobin, desmin, creatine kinase, troponins) and cytokeratins (KRT6A, 6B, 13, 14, and 78), upregulation of several neuron-specific genes [neural cell adhesion molecule 1 (NCAM-1/CD56), neurofilament (NEFH)], along with some well-characterized tumor biomarkers [carbonic anhydrase IX (CA-9), topoisomerase IIα (TOP2A), matrix metalloproteinases (MMP-7, MMP-9), CDKN2 gene (p16-INK4a), checkpoint homolog 2 (CHEK2)]. Notably, both tumors showed upregulation of the pleomorphic adenoma gene 1 (PLAG1), and in 1 case PLAG1 gene rearrangement was detected by break-apart FISH. Some vulvar tumors with morphologic and immunohistochemical characteristics of EMC may represent a molecular genetic entity separate from EMCs arising in other locations. PLAG1 gene activation appears to be involved in the development of these neoplasms.