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High-grade serous carcinoma (HGSC) is the most common and aggressive type of ovarian cancer, and it is often associated with ascites at presentation. The objective of this study was to evaluate the accuracy of cytopathology to identify immunophenotypic features of HGSC and BRCA1/2 mutations from ascites.The study included 45 patients with histologically confirmed primary HGSC and malignant ascites. Immunocytochemistry (ICC) staining for PAX8, WT1, P53, P16, and Ki-67 was performed on cytospins and cytoblocks prepared from ascites. Next-generation sequencing (NGS) was used to detect germline/somatic BRCA1/2 mutations in the ascites. Both ICC and NGS results were compared with immunohistochemistry (IHC) and NGS results from tissue blocks of primary tumor. Cronbach α and χICC/IHC results for PAX8, WT1, P53, and P16 showed good reliability between cytospins, cytoblocks, and tissue blocks (α 0.75), whereas poor reliability and significant differences were observed for Ki-67 between ascites and tissue blocks (α 0.26; p.001 [Kruskal-Wallis]). For germline BRCA1/2 mutations, 100% concordance was confirmed, but only 14% concordance was confirmed for somatic mutations.These results demonstrate that cytopathology is an accurate method for identifying immunophenotypic features of HGSC and detecting germline BRCA1/2 mutations from ascites. However, further investigation is required for assessing the proliferation activity of HGSC in ascites and for detecting somatic BRCA1/2 mutations.

Izhodišče: Ocena razširjenosti raka endometrija pred operaciji s slikovnimi metodami ni zanesljiva. Od ocene je odvisno, ali naj kirurško zdravljenje vključuje pelvično limfadenektomijo ali ne. Biopsija varovalne bezgavke lahko varno nadomesti radikalno pelvično limfadenektomijo pri bolnicah z nizkim in zmernim tveganjem za ponovitev bolezni.Metode: Od januarja 2016 do junija 2017 je bilo na Ginekološki kliniki v Ljubljani v pregled začetnih kliničnih rezultatov ob uvedbi biopsije v varovalni bezgavki v rutinsko klinično prakso vključenih 35 bolnic. Beležili smo zanesljivost histološke in ultrazvočne ocene pred operacijo ter stopnjo uspešne kirurške detekcije v varovalni bezgavki s cervikalno aplikacijo zelenila indocianin. Vsa odstranjena tkiva so bila poslana na histološko preiskavo z barvanjem po metodi hematoksilin in eozin (H&E).Rezultati: Enostranska uspešnost kirurške detekcije varovalne bezgavke je bila 85,7 % (75–93 %), obojestranska pa 80,0 % (63–92 %). Varovalna bezgavka je bila histološko pozitivna v dveh primerih. Ultrazvočna ocena invazije v miometrij je imela občutljivost 100 % (15,8–100 %) in specifičnost 78,9 % (54,4–93,9 %), ultrazvočna ocena invazije v stromo materničnega vratu pa le 33 % (0,8–90,6 %) občutljivosti in 94,4 % (72,7–99,8 %) specifičnosti. Ocena histološke stopnje diferenciacije je bila po operaciji višja (angl. upgrading) v 5,7 %, nižja (angl. downgrading) pa v 8,6 %.Zaključek: Odstranjevanje varovalne bezgavke omogoča individualnejši pristop k zdravljenju bolnic z rakom endometrija in varnejšo opustitev pelvične limfadenektomije pri bolnicah z nizkim in zmernim tveganjem za ponovitev bolezni. Za dokončno umestitev v smernice zdravljenja bodo v našem prostoru potrebne dodatne izkušnje glede izbire bolnic, sledenja kakovosti kirurške obravnave in nujna uvedba res poadrobnega histološkega pregleda – t. i. ultrastaginga (angl. ultrastaging) odstranjene varovalne bezgavke.

The aim of the study was to analyze the overall survival (OS) and progression free survival (PFS) of patients with high grade and advanced stage epithelial ovarian cancer (EOC) with at least 60 months of follow-up treated in a single gynecologic oncology institute. We compared primary debulking surgery (PDS) versus neoadjuvant chemotherapy plus interval debulking surgery (NACT + IDS) stratifying data based on residual disease with the intent to identify the rationale for therapeutic option decision and the role of laparoscopic evaluation of resectability for that intention. Patients and methods. This is observational retrospective study on consecutive patients with diagnosis of high grade and International Federation of Gynecology and Obstetrics (FIGO) stage III/IV EOC referred to our center between January 2008 and May 2012. We selected only patients with a follow-up of at least 60 months. Primary endpoint was to compare PDS versus NACT + IDS in term of progression free survival (PFS) and overall survival (OS). Secondary endpoints were PFS and OS stratifying data according to residual disease after surgery in patients receiving PDS versus NACT + IDS. Finally, through Cox hazards models, we tested the prognostic value of different variables (patient age at diagnosis, residual disease after debulking, American Society of Anesthesiologists (ASA) stage, number of adjuvantchemotherapy cycles) for predicting OS. Results. A total number of 157 patients were included in data analysis. Comparing PDS arm (108 patients) and NACT + IDS arm (49 patients) we found no significant differences in term of OS (41.3 versus 34.5 months, respectively) and PFS (17.3 versus 18.3 months, respectively). According to residual disease we found no significant differences in term of OS between NACT + IDS patients with residual disease = 0 and PDS patients with residual disease = 0 or residual disease = 1, as well as no significant differences in PFS were found comparing NACT + IDS patients with residual disease = 0 and PDS patients with residual disease = 0; contrarily, median PFS resulted significantly lower in PDS patients receiving optimal debulking (residual disease = 1) in comparison to NACT + IDS patients receiving complete debulking (residual disease = 0). PDS arm was affected by a significant higher rate of severe post-operative complications (grade 3 and 4). Diagnostic laparoscopy before surgery was significantly associated with complete debulking. Conclusions. We confirm previous findings concerning the non-superiority of NACT + IDS compared to PDS for the treatment of EOC, even if NACT + IDS treatment was associated with significant lower rate of post-operative complications. On the other hand, selecting patients for NACT + IDS, based on laparoscopic evaluation of resectabilty prolongs the PFS and does not worse the OS compared to the patients not completely debulked with PDS. Na odločitev o vrsti zdravljenja bolnikov z napredovalim melanomom lahko pomembno vpliva status mutacij v genih za BRAF, NRAS IN c-KIT. Pogostost mutacij in njihove medsebojne povezave s histološkimi značilnostmi tumorskih tkiv na slovenski populaciji do sedaj še niso bili raziskani. Bolniki in metode. Analizo smo izvedli retrospektivno. Mutacije BRAF, NRAS in c-KIT smo določili na 230 patoloških vzorcih bolnikov, ki smo jih nameravali zdraviti s sistemsko terapijo zaradi metastatskega melanoma na Onkološkem Inštitutu Ljubljana med leti 2013 in 2016. Zbrali smo histološke značilnosti primarnih tumorjev in klinične podatke bolnikov ter jih testirali na medsebojno povezanost z mutacijskim statusom. Rezultati. Povprečna starost 230 bolnikov je bila 59 let (razpon 25%85). Moških je bilo 141 (61,3 %) in žensk 89 (38,7 %). Ugotovili smo 129 (56,1 %) mutacij BRAF, 31 (13,5 %) NRAS in 3 (1,3 %) c-KIT mutacij v tkivnih vzorcih. Med 129 bolniki z mutacijami BRAF je imelo 114 (88,4 %) bolnikov mutacijo V600E in 15 (11,6 %) mutacijo V600K. Bolniki z BRAF mutacijami so bili ob diagnozi mlajši (52 v primerjavi s 59 let, p < 0,05), bolniki z NRAS mutacijami starejši (61 v primerjavi s 55 leti, p < 0,05). Število mutacij c-KIT je bilo prenizko za smiselno testiranje na medsebojne povezanosti, je pa bil eden izmed treh melanomov s c-KIT mutacijo melanom sluznice. Zaključki. Rezultati analize so v skupini slovenskih bolnikov z metastatskim melanomom odkrili visok delež mutacij BRAF ter nizek delež mutacij NRAS in c-KIT v primerjavi s predhodno objavljenimi raziskavami v Evropi in Severni Ameriki. Eden od glavnih vzrokov so specifične značilnosti naše študijske populacije, ki niso bile enake kot značilnosti celotne populacije bolnikov z melanomom.

Background The incidence of cervical cancer (CC) in Slovenia in 2011 was 13.2 per 100,000 women. The treatment of early stages of invasive cervical carcinoma involves several surgical techniques. In this article the authors would like to present a new combination of two methods which help to preserve fertility and to improve pregnancy outcome. The first procedure, radical vaginal trachelectomy (RVT), begins with laparoscopic pelvic lymphadenectomy. All suspicious lymph nodes are sent to frozen section. If those lymph nodes are negative, the procedure continues vaginally. Almost the entire cervix is removed with parametria and vaginal cuff. Permanent cerclage stitch is applied and covered with vagina on what is left of uterus. Second procedure, laparoscopic abdominal cerclage (LAC), begins with pneumoperitoneum. Mersilene tape is introduced in the abdominal cavity and placed through the visceral peritoneum at the isthmic part of the uterus with a Berci's needle. It is knotted and remains permanently. Materials and methods For the first procedure all the patients with confirmed cervical carcinoma (FIGO Stage IA₁, IA₂, and IB₁) and with the desire for fertility were recruited. For the second procedure, all the patients after RVT and after miscarriage after 14th week of gestation were recruited. Results RVT was performed in 15 patients and laparoscopic abdominal cerclage in three of them (21.5%). All three patients achieved pregnancies and after 36th weeks of gestation delivered by cesarean section (100%). Conclusions RVT alone is an indication for LAC. Considering its success, LAC should be performed before any miscarriage.

We sought to compare VeraCept (VC175), a novel nitinol intrauterine contraceptive (IUC) with 175 square-mm of copper surface area, to a copper T380S IUC.We enrolled parous women into a randomized subject-blinded comparison of VC175 and a copper T380S in a 2:1 fashion at a single clinic. The primary outcomes were total adverse events and continuation at 12 months. We also examined pain on insertion, ease of placement, expulsion, tolerability and pregnancy. Subjective ratings were on a 5-point Likert scale (0, no pain to 5, worst pain). We followed subjects through 24-month follow-up.We enrolled 300 women with 199 randomized to VC175 and 101 to the T380S. Insertion was successful in 198 subjects for VC175 and 100 for the T380S. Mean age was 25 years (range 18, 41), and median parity was 2 (range 1, 8), with 39% having only had Cesarean deliveries. No subjects developed clinical infection or reported serious adverse events. In the VC175 and T380S groups, mean pain at insertion was 1.4 and 2.4, respectively (p.01). At the 12-month primary endpoint for VC175 and T380S, respectively, continuation was 84% and 68% (p.002) with expulsions in 5.0% and 12.0% (p.05) and removal for pain/bleeding in 3.5% and 17.0% (p.01). At the 24-month visit for VC175 and T380S, respectively, continuation was 77% and 62% (p.02 by log-rank). One ectopic pregnancy was identified at the 12-month follow-up in a VC175 user. No other pregnancies were diagnosed. With 297.3 and 132.4 woman-years, pregnancy rates were 0.3 and 0.0 per 100 woman-years for VC175 and T380S, respectively.VC175 resulted in less pain at insertion, fewer expulsions and higher total continuation than the T380S, with similar contraceptive efficacy.VC175 is a promising new intrauterine copper contraceptive on a nitinol frame that warrants further clinical trials.

Onkologija, leto XXII, št. 1, junij 2018, Onkologija, Volume XXII, No. 1, June 2018

Rak materničnega vratu lahko prizadene ženske v rodnem obdobju. V stadiju IB1 je mogoče pri zdravljenju rakave bolezni ohraniti rodno funkcijo ženske z radikalno trahelektomijo in odstranitvijo medeničnih bezgavk.To je operacija, pri kateri se odstrani maternični vrat in paracervikalno tkivo (parametriji) v kombinaciji z odstranitvijo medeničnih bezgavk, telo maternice pa se ohrani. Predstavljamo primer 32-letne nulipare z rakom materničnega vratu stadija 1B1. Bolnica je bila zdravljena radikalno z ohranitvijo rodne funkcije in je po zdravljenju rodila donošenega otroka.

Between 2008 and 2010, four patients were treated for leiomyomatosis peritonealis disseminata, three were symptomatic and in one pelvic tumor was found during a routine pelvic examination. All four patients were in reproductive age and had never used any hormonal medication. Possible etiological factors were analyzed. The only common feature we found in these patients was laparoscopic myomectomy (in one additional laparoscopic supracervical hysterectomy 1 year after the first procedure) performed 3–11 years previously. Laparoscopy confirmed multiple myoma-like tumors and was converted to laparotomy in two patients; in the two patients, the tumors were removed laparoscopically. Laparoscopic myomectomy with morcellation could be an etiological factor for leiomyomatosis peritonealis disseminata. In selected cases and in experienced hands, the disease can be treated laparoscopically.

Little is known about parthenogenesis in the human ovary. What is known is related to patients with teratoma in their medical history. Ovarian surface epithelium (OSE) was often proposed as a source of ovarian stem cells with an embryonic character in the past, and was also termed "germinal epithelium." The aim of this study was to isolate putative stem cells from OSE scrapings, to set up an OSE cell culture, to follow the in vitro oogenesis and possible formation of parthenogenetic embryos in 21 postmenopausal women with no naturally present follicles and oocytes. Small round cells with a bubble-like structure and with a diameter from 2 to 4 microm were isolated from the material obtained by OSE scrapings in all women. They expressed early embryonic developmental markers such as stage-specific embryonic antigen-4 (SSEA-4) surface antigen and Oct-4, Nanog, Sox-2, and c-kit transcription factors. These cells were separated by density gradient centrifugation and grown in vitro, where they proliferated and formed embryoid body-like structures. Their markers of pluripotency such as telomerase activity were decreased during in vitro culture and they did not form teratoma after the injection into SCID mice. Some of them grew intensively and reached a diameter of approximately 20 microm after 5-7 days of culture. In the OSE cell culture, oocyte-like cells developed among them, which reached a diameter up to 95 mum, and expressed Oct-4, c-kit, VASA, and ZP2 transcription markers after 20 days of culture. Some of them expressed a zona pellucida-like structure and rarely germinal vesicle- and polar body-like structures. At the same time, parthenogenetic blastocyst-like structures developed, which expressed transcription markers Oct-4, Sox-2, and Nanog and were normal for chromosomes X, Y, 13, 16, 18, 21, and 22. In conclusion, the discovered cells expressed embryonic stem cell markers, gave rise to embryoid body-, oocyte-, and blastocyst-like structures, and might be involved in the human reproduction and formation of ovarian tumors.

The authors present their experience with the detection of the sentinel node, which is the first node to receive drainage from a malignant tumor, in patients with cervical cancer. Fifty women who were scheduled to undergo radical abdominal hysterectomy and systematic pelvic lymphadenectomy for treatment of stage IA2 to IIA primary carcinoma of cervix from January 2003 to January 2005 served as study subjects. Sentinel lymph node (SNL) mapping was done with an intracervical injection of 4 mL methylene blue. Pelvic lymph nodes appeared blue 15 minutes after dye injection and remained colored for 70 minutes. A 25-gauge standard needle was used to slowly, under constant pressure, inject the dye into the cervix to a depth of 5 to 10 mm at 3, 6, 9, and 12 o'clock positions, taking care to avoid injecting into the tumor itself. The abdomen was then opened, and the right retroperitoneal space on the right pelvis was opened. Using blunt dissection, the avascular paravesical and pararectal spaces were developed. The blue lymphatics could then be followed to the first node with obvious or faint dye uptake. This sentinel node was dissected and removed. The procedure was repeated on the left side before proceeding with lymphadenectomy. After the SNLs were removed on both sides, bilateral pelvic lymphadenectomy followed by radical hysterectomy was performed in all patients. The technique was considered to have failed when no sentinel node was identified or when there was inadequate injection of the dye. If the SNL was negative on histology, but other nodes in the drainage area were positive, the outcome was considered to be a false-negative. Sentinel nodes were considered positive when they contained macrometastases, micrometastases, or isolated tumor cells. Sentinel nodes were processed in the routine fashion with hematoxylin & eosin staining, but negative sentinel nodes underwent additional processing using serial step sections and wide-spectrum cytokeratin immunohistochemical analysis. The mean age of participants was 45.9 years. The most common preoperative diagnosis was clinical stage IB1 cervical cancer. Two women had microinvasive cancer stage IA2. Seventeen patients underwent a cone biopsy before surgery. Residual carcinoma in situ was diagnosed in 18% of these patients, one third of whom had lesions larger than 2 cm. A total of 1093 lymph nodes were retrieved (mean, 21.9 per patient). Eighty-six sentinel nodes were identified in 45 patients (detection rate, 90%). Forty SNLs were on the right side in 34 patients, and 46 SNLs were on the left side in 38 patients. Twenty-five women (50%) had two SNLs identified and 18 (26%) had one SNL identified. In five women, there were three SNLs, and two patients had four sentinel nodes. No sentinel nodes were identified in five patients (failure rate, 10%). Inadequate injection technique with intraperitoneal spillage accounted for two of the failures. A third was attributed to injection of the methylene blue into the tumor in a woman with bulky cervical cancer. After this, patients with bulky tumors were excluded from the study. The external iliac and obturator areas were the most common sites of SNL (55% and 38%, respectively). SNLs were similarly distributed on both sides of the pelvis. In this series, sentinel nodes detected in the common iliac area (n = 5) typically were the only positive nodes found on the same side. There was one parametrial SNL, but no dye could be seen beyond it. Twenty-five positive nodes were identified in 10 patients (20%), including seven with unilateral involvement and three with bilateral spread. Thirteen of the 25 positive nodes were identified as sentinel nodes and were found in nine of the 10 patients. In five patients, the SNL was the only positive node detected. The one woman with a false-negative SNL had stage IB 1 squamous cervical cancer and one positive right parametrial lymph node and 35 negative pelvic lymph nodes. Bilateral SNLs identified in this patient were negative by serial step and wide-spectrum cytokeratin immunohistochemical analysis.

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 μm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.

The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to thein vitrodevelopment of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched.

Background: It has already been found that very small embyronic-like stem cells (VSELs) are present in adult human tissues and organs. The aim of this study was to find if there exists any similar population of cells in cell cultures of reproductive tissues and embryonic stem cells, and if these cells have any relation to pluripotency and germinal lineage. Methods and results: Here we report that a population of small SSEA-4-positive cells with diameters of up to 4 μm was isolated by fluorescence-activated cell sorting (FACS) from the human ovarian cell cultures after enzymatic degradation of adult cortex tissues. These small cells – putative ovarian stem cells – were also observed during cell culturing of up to 6 months and more. In general, small putative ovarian stem cells, isolated by FACS, showed a relatively low gene expression profile when compared to human embryonic stem cells (hESCs) and human adult fibroblasts; this may reflect the quiescent state of these cells. In spite of that, small putative ovarian stem cells expressed several genes related to primordial germ cells (PGCs), pluripotency and germinal lineage, including VASA. The PGC-related gene PRDM1 was strongly expressed in small putative ovarian stem cells; in both hESCs and fibroblasts it was significantly down-regulated. In addition, putative ovarian stem cells expressed other PGC-related genes, such as PRDM14 and DPPA3. Most of the pluripotency and germinal lineage-related genes were up-regulated in hESCs (except VASA). When compared to fibroblasts, there were several pluripotency-related genes, which were up-regulated in small putative ovarian stem cells. Similar populations of small cells were also isolated by FACS from human testicular and hESC cultures. Conclusions: Our results confirm the potential embryonic-like character of small putative stem cells isolated from human adult ovaries and their possible relation to germinal lineage.

There have been some proposals that stem cells exist in the ovarian surface epithelium (OSE) of the adult human ovary; however, no direct evidence of such cells has been given until now. The aim of this study was to isolate the putative ovarian stem cells (OSCs) from the OSE layer in women with no naturally present oocytes and follicles--20 postmenopausal women and five women with premature ovarian failure. Small round cells with a bubble-like structure and diameters from 2 to 4 microm were isolated from the material obtained by OSE scraping. They expressed early embryonic developmental markers such as stage-specific embryonic antigen-4 and Oct-4, Nanog, Sox-2, and c-kit transcription markers, and they displayed prominent c-kit immunohistochemical staining. These cells were separated by density gradient centrifugation and grown in vitro, where they proliferated. Some of them grew intensively and reached a diameter of approximately 20 microm after 5-7 days. In the OSE cell culture, oocyte-like cells developed, which reached a diameter of up to 95 microm and expressed Oct-4A, Oct-4B, c-kit, VASA, and ZP2 transcription markers, corresponding to early oocytes. They did not express SCP3 meiotic marker. In conclusion, the discovered cells are proposed to represent the adult OSCs with the expression of embryonic stem cell markers. The expression of germ lineage marker c-kit points toward their primordial germ cell ancestry. A new term "embryonic-like stem cells of the adult" is proposed for embryonic-like stem cells that might persist in various tissues and organs of adults. These findings could be used for further studies aimed at the autologous treatment of ovarian infertility and degenerative diseases.

Introduction The presence of oocytes in the ovarian surface epithelium has already been confirmed in the fetal ovaries. We report the presence of SSEA-4, SOX-2, VASA and ZP2-positive primitive oocyte-like cells in the adult ovarian surface epithelium of a patient with serous papillary adenocarcinoma. Case presentation Ovarian tissue was surgically retrieved from a 67-year old patient. Histological analysis revealed serous papillary adenocarcinoma. A proportion of ovarian cortex sections was deparaffinized and immunohistochemically stained for the expression of markers of pluripotency SSEA-4 and SOX-2 and oocyte-specific markers VASA and ZP2. The analysis confirmed the presence of round, SSEA-4, SOX-2, VASA and ZP2-positive primitive oocyte-like cells in the ovarian surface epithelium. These cells were possibly related to the necrotic malignant tissue. Conclusion Primitive oocyte-like cells present in the adult ovarian surface epithelium persisting probably from the fetal period of life or developed from putative stem cells are a pathological condition which is not observed in healthy adult ovaries, and might be related to serous papillary adenocarcinoma manifestation in the adult ovarian surface epithelium. This observation needs attention to be further investigated.