[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.

[P16NIK4a expression in premalignant cervical lesions]. / Ekspresija stanicnog proteina p16INK4a kod premalignih promjena vrata maternice.

Increased expression of viral E6 and E7 oncoproteins within the host cells results in an increase in cellular protein p16INK4a expression. That increase may serve as a marker for dysplastic and neoplastic cells of the uterine cervical epithelium. The aim of this study is to assess the p16INK4a protein expression in different stages of cytological abnormality in correlation with the proven high oncogenic risk human papillomavirus infection in order to demonstrate its value as the diagnostic marker. The study included cervical smear samples of 371 patient in whom the viral typization was done. In 171 patient, during their regular gynaecological examination, along with conventional Pap smear sampling an additional smear was taken. Two hundred cervix brush (Rovers Medical DevicesOss, the Netherlands) samples were obtained and analyzed by the LBC method and the ThinPrep2000 machine. All samples were analyzed cytologically, classified according to the Bethesda system, and immunostained with the p16INK4a-specific monoclonal antibody E6H4 (MTM Laboratories, Heidelberg, Germany). A significant difference is seen in p16 positivity between the cytological diagnosis of a high grade cervical squamous intraepithelial lesion and the group with mild dysplasia (chi2=146,48; D.F.=4; p<0,01) while most of the mild dysplastic lesions were p16 negative. In cases of p16 positive mild dysplastic lesions, that positivity is the result of viral integration into the host genome, which would imply lesions at high risk of further progression. The potential and value of p16INK4a protein not only as a prognostic, but also as a diagnostic factor is proved in this way. Reproducibility of the p16 staining technique renders it suitable for follow-up monitoring as well as for comparison of the cytological results.

[Fine needle aspirate of lymph node as the analytical sample for immunophenotyping]. / Punktat limfnog cvora kao analiticki uzorak za fenotipizaciju stanica.

In modern clinical laboratory routine, cell analysis by flow cytometry means help in setting up the diagnosis by determination of B-lymphocyte clonality and thus separation of benign and malignant lymphoproliferative diseases. The aim of this study was to assess the value of cytologic diagnosis and adequacy of the material obtained by fine needle aspiration (FNA) of lymph nodes for flow cytometry analysis in cases of benign lesions and primary malignant lesions of lymph nodes. In addition, the aim was to determine B-lymphocyte clonality in different groups of benign and malignant lymph node lesions. The study was based on medical documentation, cytologic smears of FNA lymph node samples and results of flow cytometry immunophenotyping. A total of 239 patients were included over a one-year period. Patients were classified according to cytologic findings in the groups of non-Hodgkin's lymphoma of B cell origin (55%), benign lymphoproliferative disease (22%), undefined group of monomorphic population of lymphatic cells (16%), and the rest in the group of non-Hodgkin's non B cell origin. Study results showed FNA to be an appropriate method for obtaining sufficient numbers of cells for analysis by flow cytometry because there was no inadequate samples in our study group. In some cases of monomorphic lymphoid cell population, cytologic diagnosis was limited to small cell lymphomas, so determining the clonality by flow cytometry is crucial in separating malignant from benign lymphoproliferative disease. It is concluded that FNA associated with the flow cytometry method is a simple and safe method in the diagnosis of lymphoproliferative disease.

[Causes and frequency of unsatisfactory cervicovaginal smears]. / Nezadovoljavajuci uzorci i njihova ucestalost u cervikovaginalnim razmazima.

Inadequate Pap smear by definition is a specimen in which detection of cervical epithelial abnormality is impossible or uncertain. This causes poorer detection of intraepithelial lesions of a mild and more severe grade, including the possible false-negative diagnosis. Sample adequacy is most crucial in the evaluation of the finding, alerting the gynecologist to the limitations of the finding, its possible inaccuracy, and need to repeat the examination in order to obtain as precise results as possible. The aim of the study was to establish the frequency and reasons of unsatisfactory cervicovaginal smear samples in the course of one year, during which 1594 of 12,242 conventionally obtained cervical samples were sorted out as inadequate. These were reassessed with respect to their adequacy. Eight percent of the smears in which the evaluation of cell abnormality failed due to sample inadequacy were identified and these smears were repeated and analyzed for adequacy and presence of abnormality. The most common reasons included insufficient endocervical epithelial cells, excessive smear thickness, cells obscured with numerous inflammatory elements and erythrocytes, and sample inadequacy due to the presence of foreign material, poor fixation or staining. Inadequate equipment, insufficient material for cytologic analysis, and poor preparation technique may lead to failure to observe abnormality and errors in microscopic analysis. This implies each team member's responsibility for the accuracy of the result as well for the assurance of specimen adequacy. Reduction in the frequency of the reasons mentioned above is possible if internal control, performance quality monitoring and continuing education of each team member are conducted on a regular basis. The necessity to repeat sampling adds to the cost of health care with no considerable increase in the detection rate of epithelial abnormalities, inadequate specimens being the most common cause of false-negative cytologic findings.

[Clear cell carcinoma of the endometrium confined to atrophic endometrial polyp--case report]. / Endometralni karcinom svijetlih stanica u atroficnom endometralnom polipu--prikaz bolesnice.

Clear cell carcinoma is a rare form of endometrial carcinoma inline type II estrogen-independent. It accounts for only 1% to 5.5% of all endometrial carcinomas. It is usually detected in postmenopausal women, who are older than those with endometrioid carcinoma. Clear cell carcinoma may exhibit solid, papillary, tubular and cystic patterns. The malignant cells are characterized by a moderate amount of clear or foamy cytoplasm; however, its recognition as a clear cell type of cancer cytologically is not often possible. The tumor is usually poorly differentiated, tends to be high grade, with deep myometrial invasion, lymphovascular space invasion, and metastasis in pelvic lymph node. That is why this tumor has a poor prognosis, as in our case of a 72-year-old woman with symptoms of deficient uterine bleeding. In a period of two months, two suction endometrial curettages were performed. The material obtained by endometrial curettage for histopathologic verification was very scanty. The assessment described an inactive endometrium with degenerative epithelial cells in scanty necrotic background. Direct endometrial samples with uterobrush yielded a finding of atypical epithelial cells of open etiology with dense chromatin and prominent nucleoli, also with foam cell histiocytes and old blood in the background. Cytologic diagnosis of intraepithelial lesion, possible adenocarcinoma was made. Ultrasonography of the uterus was suspect of neoplasm. After surgery, the pathologic diagnosis of endometrial clear cell carcinoma was made. It was a rare case of aggressive type of endometrial carcinoma confined to an atrophic endometrial polyp. In conclusion, we might say that direct endometrial sample can provide accurate diagnosis of endometrial tumor, especially when the endometrial tumor is a small one, thus saving time for diagnostic testing. This is based on the potential of cytology to make malignant diagnosis possible even with a few tumor cells on the slide.

[Preoperative diagnosis of fallopian tube carcinoma by cytology]. / Preoperativna citoloska dijagnoza karcinoma jajovoda--prikaz bolesnice.

Carcinoma of the fallopian tube is a rare, extremely aggressive neoplasm that is uncommonly diagnosed in early stages of the disease. We present a case of a 56-year-old nulliparous patient with tubal carcinoma stage Ic (FIGO). Preoperatively, the only suspect finding were atypical glandular cells of extrauterine origin presented on a routine Papanicolaou smear. Histopathologic finding of D&C implicated the same suspicion of extrauterine genital malignancy. After hysterectomy with bilateral adnexectomy and omentectomy, histopathology confirmed stage I primary adenocarcinoma of the right fallopian tube (alveomedullary type) with positive intraoperative cytology of Douglas pouch lavage. As the patient had bilateral occlusion of the tubes, we presumed that tumor cells were leaking through the uterus into the vagina, and we were luckily alerted within the pathological cervical smear to the possible presence of a malignancy in the abdomen. The patient received 6 courses of cyclophosphamide and cisplatin chemotherapy postoperatively. Two years after the diagnosis, the patient is healthy, without a trace of primary malignant disease. Cytology undoubtedly plays a vital role as a screening tool in the detection of premalignant and malignant diseases of the female genital tract, particularly lesions in the uterine cervix. However, its role in clinical staging of gynecologic diseases should not be underestimated either, due to its ability to detect malignant epithelial cells in peritoneal samples as well as for its detecting and diagnostic value in other malignant gynecologic diseases.

[Primary ovarian malignant mixed mesodermal tumor (MMMT) as a second primary tumor in a patient with invasive breast carcinoma--case report]. / Maligni mijesani mezodermalni tumor jajnika (MMMT)--drugi primarni tumor u bolesnice s invazivnim karcinomom dojke.

Malignant mixed mesodermal tumor (MMMT) of the ovary is a rare aggressive tumor that consists of an epithelial (carcinoma) and a stromal (sarcoma) component. MMMT accounts for less than 2% of ovarian cancers and has a very poor prognosis. We present a case and difficulties of diagnosing an ovarian MMMT in a postmenopausal woman with a history of invasive breast carcinoma treated postoperatively with radiotherapy and tamoxifen. A 52-year-old patient presented with unilateral ovarian tumor and moderately elevated CA125 (107 U/mL) and underwent laparotomy. Fine needle aspiration of the ovary and ascites for cytologic analysis, and tumor biopsy for histopathology were performed intraoperatively. Intraoperative cytologic sample showed necrotic background with rare single malignant cells with pale, abundant cytoplasm and conspicuous nucleoli suggesting clear cell carcinoma. Ascites sample showed inflammatory and reactive background with suspected papillary formations mimicking adenocarcinoma. Postoperatively, cytochemical PAS staining and immunocytologic staining with epithelial antigen (EA), cytokeratin (CK)7 and vimentin showed EA and PAS positivity for ovarian tumor, and EA and CK7 for ascites, suggesting a clear cell carcinoma. Histology revealed ovarian clear cell carcinoma. Three months later, the patient underwent hemicolectomy because of tumors on the right large bowel serosa with intraoperative morphological finding of metastatic malignant tumor without other specific features. Postoperative morphological analysis and immunohistochemical staining of the tumor revealed two malignant components, epithelial and stromal one. Repeat histologic analysis of the ovarian tumor confirmed ovarian MMMT (with a clear cell carcinoma component). Other studies of breast cancer emphasize that patients with invasive breast cancer and mutations of BRCA1 and BRCA2 genes are at an increased risk of primary ovarian cancer. Our study confirmed it and suggested considering a second primary malignant tumor of ovarian origin in patients with a history of breast carcinoma, postoperatively treated with radiotherapy and tamoxifen. Although rare, second primary ovarian tumors may present as MMMT.

Vulvar Paget's disease--a case report.

Vulvar Morbus Paget (MP) represents a rare intraepithelial adenocarcinoma. It accounts for less than 1% of all vulvar neoplasia and usually appears in postmenopausal women. Histologically it is analogous to Paget's disease of the breast. The most common clinical symptom is pruritus. The lesion appears as an erythematous or as an eczematous lesion with islands of hyperkeratosis. Occasionally, single anaplastic Paget's cells can be found on the vulvar smears which make cytological diagnosis of the disease possible. However, the disease can be diagnosed only by biopsy. We present a case of 49-year old woman with vulvar symptoms of pruritus, who had liver and kidney transplantation two years ago. During the standard gynecological examination the vulvar smear was taken for cytological evaluation. The smear was scanty, with inflammatory background, overloaded with squamae. There were two types of cells: dysplastic squamous cells from lower layer of the epithelium and the single, anaplastic cells with a high nuclear:cytoplasmic ratio who possessed eccentric, large nucleus. Nucleoli were rare. Cytoplasm varied from pale and delicate to densely basophilic. Accordingly, cytological diagnosis vulvar intraepithelial neoplasia (VIN III) with differential diagnosis of vulvar Paget's disease was made. The pathological verification supported the diagnosis of MP and an immunohistochemistry panel confirmed type III of Paget's disease and an evaluation of bladder was suggested.

Estimating clinical outcome of HPV induced cervical lesions by combination of capsid protein L1 and p16INK4a protein detection.

The aim of this study was to investigate whether is possible to predict clinical outcome of cervical lesion by immunoassaying performed on cervical smears. During the two year study period the cervical smears of 81 patients were collected. All patients were tested for human papillomavirus (HPV) infections using Amplycor HPV test. Sixty-six of them were tested as positive for high risk types (hrHPV) and squamous intraepithelial lesion, and in those patients repeated cervical smears were taken every six months. The rest were hrHPV negative patients with normal smears which were used as a negative control in immunoassays with HPV L1 and p16INK4a antibodies. The results of p16INK4a staining in 66 hrHPV positive patients showed impairment of the cervical lesion in 22 (33.3%) and unchanged cytological finding in 21 (31.9%) p16INK4a positive patients, respectively, while improving of cytological finding was seen only in three (4.5%) p16LNK4a positive patients. On the contrary, impairment of cytological finding was not seen in p16INK4a negative patients, while in 17 out of 20 patients from that group improving or normalisation of cytological finding were detected (p < 0.01). Correlation between L1/p16 pattern and cytological finding showed that only in L1-/p16+ cervical lesions was detected impairment of cytological finding during the study period. In L1 +/p16+ group the cytological finding was the same during the follow up in all 11 patients, while in L1 +/p16- group in most patients (9/11) downgrading or normalisation of Pap test were detected. The usage of p16 and HPV L1 markers can be useful in estimation of biologic potentiality and clinical outcome of cervical lesions.

Diagnostic methods and techniques in preventing cervical carcinoma. Part I: Conventional cytology and new cytological methods.

Cancer of the cervix is one of the most predictable and preventable types of cancer, however, it is still one of the most common malignancies. Due to a lack of information available to women about the causes of the disease, accessibility of screening programs, and limitations to the existing screening techniques, cervical cancer is the second most common type of cancer in women worldwide. Detection and follow-up of pre-cancer stages of the disease are based on the Pap test, which is now well established as a basic method of secondary prevention. Relative low sensitivity of the Pap test has initiated the development of additional technologies and methods towards enhanced screening quality and error elimination not only in the process of sample taking and analysis but also in screening and interpretation. Immunocytochemical methods and liquid based cytology are the new diagnostics possibilities in secondary prevention. In order to decrease morbidity, thus mortality too, it is necessary that the primary prevention (vaccination) be also implemented.

Evaluation of p16INK4a in cervical lesion of premenopausal and postmenopausal women.

Pap smears of postmenopausal women are often misdiagnosed because of the difficulty in distinguishing atrophic epithelial cells groups only by morphological criteria. In this study we investigated the diagnostic application of immunocytochemical staining of p16INK4a on conventional Pap smear. A total of 137 cervical specimens were enrolled in this study, of which 77 and 60 cervical smears were taken from premenopausal and postmenopausal women, respectively. Two cervical smears were taken simultaneously in 68 women, one for conventional cytology and the other for immunostaining. Additional 69 cervical smears were taken from the archive, decolorized and then used for immunostaining. In premenopausal women 1 out of 14 (7.1%) with negative cytology, 7 out of 24 (29.2%) with low grade squamous intra-epithelial lesion (LSIL), all 35 (100%) with high grade squamous intraepithelial lesion (HSIL) and all 4 (100%) with squamous cell carcinoma (confirmed by histopathology) had positive staining to p16INK4a. In postmenopausal women p16INK4a positivity was observed in 4 out of 7 (57.1%) cases of LSIL, 12 out of 14 (85.7%) cases of HSIL and all 4 out of 5 (80%) different cases of carcinoma (1 cervical adenosquamous carcinoma and 3 cervical squamous cell carcinoma in situ confirmed by histopathology), but none of 34 smears with normal cytology. Twenty smears with normal cytology chosen for the negative control in this study were from the group of postmenopausal women and were as expected negative for p16INK4a immunostaining. In the group of postmenopausal women, 16 out of 60 (26.7%) cases the cytological diagnosis was established on the basis of pl6lNK4a immunostaining as being HSIL. From our preliminary study on a limited number of samples, we can however conclude that pl6INK4a immunostaining is a very useful tool for cytological diagnosis enabling to distinguish HSIL from normal, reactive or inflammatory changes.