Distribution of human papillomavirus genotypes in women with high-grade cervical intraepithelial lesions and cervical carcinoma and analysis of human papillomavirus-16 genomic variants.

AIM: To analyze the distribution of high-risk human papillomavirus (HR-HPV) genotypes and the diversity of HPV-16 genomic variants in Croatian women with high-grade squamous intraepithelial lesions (HSIL) and cervical carcinoma. METHODS: Tissue biopsy specimens were obtained from 324 women with histopathologically confirmed HSIL or cervical carcinoma, 5 women with low-grade SIL, and 49 women with negative histopathology. HR-HPV DNA was detected with Ampliquality HPV-type nucleic-acid hybridization assay, which identifies 29 different HPV genotypes. HPV-16 genomic variants were analyzed by an in-house sequencing. RESULTS: The most common HPV type in women with HSIL was HPV-16, detected in 127/219 (57.9%) specimens. HPV-16 was also the dominant type in squamous cell cervical carcinoma (46/69 or 66.7%) and in adenocarcinoma (18/36 or 50.0%). Out of 378 patients, 360 had HR-HPV (282 single infections and 79 multiple infections), 3 (0.8%) patients had low-risk HPV, and 15 (4%) tested negative. HPV-16 variants were determined in 130 HPV-16 positive specimens, including 74 HSIL and 46 carcinoma specimens. In HSIL specimens, 41 distinct variants were found, 98.6% belonging to the European branch and 1.4% belonging to the African branch. In cervical carcinoma specimens, 95% isolates grouped in 41 variants belonging to the European branch, one isolate (2.5%) belonged to the North American, and one (2.5%) to the Asian-American branch. CONCLUSION: HPV-16, mainly belonging to the European branch, was the most frequent HPV genotype in women from Croatia with histologically confirmed HSIL and cervical cancer.

D variants in the population of D-negative blood donors in the north-eastern region of Croatia.

OBJECTIVES: The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. BACKGROUND: Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. MATERIAL/METHODS: A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. RESULTS: RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. CONCLUSION: Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

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.

Methylated Host Cell Gene Promoters and Human Papillomavirus Type 16 and 18 Predicting Cervical Lesions and Cancer.

Change in the host and/or human papillomavirus (HPV) DNA methylation profile is probably one of the main factors responsible for the malignant progression of cervical lesions to cancer. To investigate those changes we studied 173 cervical samples with different grades of cervical lesion, from normal to cervical cancer. The methylation status of nine cellular gene promoters, CCNA1, CDH1, C13ORF18, DAPK1, HIC1, RARß2, hTERT1, hTERT2 and TWIST1, was investigated by Methylation Specific Polymerase Chain Reaction (MSP). The methylation of HPV18 L1-gene was also investigated by MSP, while the methylated cytosines within four regions, L1, 5'LCR, enhancer, and promoter of the HPV16 genome covering 19 CpG sites were evaluated by bisulfite sequencing. Statistically significant methylation biomarkers distinguishing between cervical precursor lesions from normal cervix were primarily C13ORF18 and secondly CCNA1, and those distinguishing cervical cancer from normal or cervical precursor lesions were CCNA1, C13ORF18, hTERT1, hTERT2 and TWIST1. In addition, the methylation analysis of individual CpG sites of the HPV16 genome in different sample groups, notably the 7455 and 7694 sites, proved to be more important than the overall methylation frequency. The majority of HPV18 positive samples contained both methylated and unmethylated L1 gene, and samples with L1-gene methylated forms alone had better prognosis when correlated with the host cell gene promoters' methylation profiles. In conclusion, both cellular and viral methylation biomarkers should be used for monitoring cervical lesion progression to prevent invasive cervical cancer.

[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.

Breast and gynecological cancers in Croatia, 1988-2008.

AIM: To analyze and interpret incidence and mortality trends of breast and ovarian cancers and incidence trends of cervical and endometrial cancers in Croatia for the period 1988-2008. METHODS: Incidence data were obtained from the Croatian National Cancer Registry. Themortality data were obtained from the World Health Organization (WHO) mortality database. Trends of incidence and mortality were analysed by joinpoint regression analysis. RESULTS: Joinpoint analysis showed an increase in the incidence of breast cancer with estimated annual percent of change (EAPC) of 2.6% (95% confidence interval [CI], 1.9 to 3.4). The mortality rate was stable, with the EAPC of 0.3%. Endometrial cancer showed an increasing incidence trend, with EAPC of 0.8% (95% CI, 0.2 to 1.4), while cervical cancer showed a decreasing incidence trend, with EAPC of -1.0 (95% CI, -1.6 to -0.4). Ovarian cancer incidence showed three trends, but the average annual percent change (AAPC) for the overall period was not significant, with a stable trend of 0.1%. Ovarian cancer mortality was increasing since 1992, with EAPC of 1.2% (95% CI, 0.4 to 1.9), while the trend for overall period was stable with AAPC 0.1%. CONCLUSION: Incidence trends of breast, endometrial, and ovarian cancers in Croatia 1988-2008 are similar to the trends observed in most of the European countries, while the modest decline in cervical cancer incidence and lack of decline in breast cancer mortality suggest suboptimal cancer prevention and control.

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.

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.

Cervical cancer as a public health issue--what next?

Cervical cancer is the second most common cancer in women worldwide. There are about 60,000 newly detected cases and 30,000 deaths annually in Europe, with the highest incidence reported from Eastern Europe countries. According to data from the National Institute of Public Health, in Croatia the incidence of cervical cancer was 14.9/100,000 in 2006, ranking eighth most common malignancy in women. Croatia has a lower incidence of the disease compared to many countries of Central and Southeast Europe. A large study carried out in 1995 by the International Agency for Research on Cancer, which included cervical cancer material collected from 22 countries all over the world revealed HPV genome in 99.7% of cases. Efficient methods of cervical cancer detection and screening methods for identification of precancerous lesions (conventional Pap smear) are available. Cervical cancer prevention programs should include education (of health care providers and women), stressing the benefits of screening, the age of the peak cervical cancer incidence, and the signs and symptoms of precancerous lesions and invasive disease. The aim of screening actions is to detect precancerous lesions that may lead to cancer if left untreated. Screening can only be effective if there is a well-organized system of follow up, diagnosis and treatment. Cervical cytology, or Papanicolaou (Pap) testing, has for decades been a cornerstone of cervical cancer screening. According to recent guidelines issued by the World Health Organization Regional Office for Europe, the primary task of the public health system is the introduction of secondary prevention through properly organized screening programs. Launching the national immunization program is only possible in the countries with well-organized secondary prevention programs and in those that can afford it.

Successful laparoscopic bipolar coagulation of a large arteriovenous malformation due to invasive trophoblastic disease: a case report.

We report the case of an acquired large arteriovenous malformation due to invasive gestational trophoblastic tumor that was treated successfully with laparoscopic surgery. After 4 cycles of methotrexate chemotherapy, a vascular tangle (volume, 28 cm(3)) was noted that emerged from the right uterine horn, invading the broad ligament adjacent to the uterine artery. Doppler ultrasonography along with magnetic resonance arteriography confirmed the diagnosis. The location, size and relation of this arteriovenous malformation to the uterine vasculature demanded urgent intervention. Laparoscopy was performed, and bipolar coagulation of the ovarian and uterine artery feeding branches was achieved after surgical resection of the tumor. The defect in the uterine wall with an intact uterine cavity was reconstructed using sutures. There were no intraoperative or postoperative complications. The patient underwent chemotherapy, and at 2-month follow-up was cured and has since had regular menstrual cycles.

Ovarian cancer: PCD and brain metastases.

Paraneoplastic cerebellar degeneration (PCD), the one of the most common paraneoplastic syndromes, refer to clinical disorders associated mostly with lung, ovarian and breast cancer, but not directly caused by the cancer or its metastases. Pathologic finding is an extensive loss of Purkinje cells in the cerebellum. Immunohistochemically, the auto-antibodies on the Purkinje cells had been detected. Clinically, PCD is characterized by sub-acutely evolving pancerebellar symptoms. Neurological dysfunction may appear before the detection of the underlying cancer. Therefore, the surgical exploration is necessary for the final diagnosis. The patient undergoes specific therapy. Soon, neurological status of the patient gets irreparable worse. Death come usually 2-3 years after the first symptoms of the PCD occurs. Case of a 63-years old woman with PCD as the first evidence of her cancer is reported. The patient developed brain metastases and died almost 3 years after the first symptoms of PCD occur

[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.

Three-dimensional ultrasonography and power Doppler in ovarian cancer screening of asymptomatic peri- and postmenopausal women.

AIM: To determine whether introducing three-dimensional (3D) ultrasonography with power Doppler facilities as a secondary screening test, preceded by annual transvaginal grayscale ultrasonography (TVUS) (followed by transvaginal color Doppler (TVCD) in selected cases) as a primary screening test for ovarian cancer improves the accuracy of ovarian cancer screening studies. METHODS: Annual TVUS was performed on 3,201 peri- and postmenopausal asymptomatic women aged > or =50 years from March 1, 2001 to June 30, 2003. Cystic ovarian lesions in perimenopausal women were routinely reevaluated by TVUS and TVCD at 4-6 week intervals to avoid unnecessary surgical intervention for physiological cysts. Any multiloculated, complex or solid ovarian mass, as well as persistently cystic mass >5 cm in diameter, in which the echo architecture and/or blood flow pattern was not highly suggestive of a benign histology, was categorized malignant. In these cases, TVUS and TVCD findings were obtained in no more than 2 weeks and supplemented by secondary screening, including 3D ultrasonography, and 3D power Doppler, in combination with serum CA 125 determination. After detailed ultrasonographic examination, surgical removal of the tumor and pathohistological classification were completed. RESULTS: Twenty-five patients (0.8%) with persisting ultrasonographic abnormalities after primary and secondary screening underwent surgery to remove the ovarian tumor. Five epithelial ovarian cancers were detected: 3 stage IA, 1 stage IB, and 1 stage IC. Three stage I patients had a palpable abnormality on clinical examination. Furthermore, in three patients with stage I disease, CA 125 serum value was elevated (> or =35 U/mL). Three-dimensional ultrasonography and power Doppler, as well as TVUS findings were indicative of malignancy in all 5 patients with stage I ovarian cancer, whereas TVCD finding was false-negative in 2 patients with stage I disease. Screening test (primary+secondary screening) had the sensitivity of 100%, specificity of 99.4%, positive predictive value of 20%, and negative predictive value of 100%. CONCLUSION: Application of 3D ultrasonography and power Doppler imaging in patients with "positive" standard ultrasound tests (annual TVUS, followed by TVCD in selected cases) represents a novel approach for the early and accurate detection of ovarian cancer through screening.