Umit Ince

The magnitude of gonadotoxicity of chemotherapy drugs on ovarian follicles and granulosa cells varies depending upon the category of the drugs and the type of granulosa cells

STUDY QUESTION Do different chemotherapy drugs exert the same magnitude of cytotoxicity on dormant primordial follicles and the growing follicle fraction in the ovary in vivo and on mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro? SUMMARY ANSWER Cyclophosphamide (alkylating agent) and cisplatin (alkylating like) impacted both primordial and pre-antral/antral follicles and both mitotic and non-mitotic granulosa cells, whereas the anti-metabolite cancer drug gemcitabine was detrimental only to pre-antral/antral follicles and mitotic non-luteinized granulosa cells. WHAT IS KNOWN ALREADY It is already known that anti-metabolite cancer drugs are less detrimental to the ovary than alkylating and alkylating like agents, such as cyclophosphamide and cisplatin. This assumption is largely based on the results of clinical reports showing lower rates of amenorrhea in women receiving anti-metabolite agent-based regimens compared with those treated with the protocols containing an alkylating drug or a platinum compound. But a quantitative comparison of gonadotoxicity with a histomorphometric proof of evidence has not been available for many chemotherapy drugs. Therefore, we combined in this study in vivo and in vitro models of human and rat origin that allows a comparative analysis of the impact of different chemotherapy agents on the ovary and granulosa cells using real-time quantitative cell indices, histomorphometry, steroidogenesis assays, and DNA damage and cell death/viability markers. We also aimed to investigate if there is a difference between mitotic and non-mitotic granulosa cells in terms of their sensitivity to the cytotoxic actions of chemotherapy drugs with different mechanisms of action. This issue has not been addressed previously. STUDY DESIGN, SIZE, DURATION This translational research study involved in vivo analyses of ovaries in rats and in vitro analyses of granulosa cells of human and rat origin. PARTICIPANTS/MATERIALS, SETTING, METHODS For the in vivo assays, 54 4- to 6-week old Sprague-Dawley young female rats were randomly allocated into four groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. MAIN RESULTS AND THE ROLE OF CHANCE Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. LIMITATIONS, REASONS FOR CAUTION This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. WIDER IMPLICATIONS OF THE FINDINGS These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells.

Clinical importance of “low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H)” terminology for cervical smears: 5-year analysis of the positive predictive value of LSIL-H compared with ASC-H, LSIL, and HSIL in the detection of high-grade cervical lesions with a review of the literature

OBJECTIVE We compared follow-up biopsy findings and positive predictive values (PPVs) for cervical intraepithelial neoplasia 2 or worse (CIN 2+) in cases that were cytologically interpreted as low-grade squamous intraepithelial lesions (LSIL); high-grade squamous intraepithelial lesions (HSIL); LSIL, cannot exclude HSIL (LSIL-H); and atypical squamous cells, cannot exclude HSIL (ASC-H) during a 5-year period to evaluate the clinical significance of LSIL-H as a distinct cytological category. METHODS All Pap tests with a diagnosis of LSIL-H, ASC-H, LSIL, and HSIL (January 1, 2004-July 20, 2009) were retrieved from our computer database. PPVs of cytological diagnostic categories for detecting CIN 2+ were compared. RESULTS Of all Pap tests (n=163,315), 1713 cases that had histological confirmation were included in the study. The LSIL-H diagnosis represented only 0.23% (n=387) of all Pap tests and 9.3% of all cytological SILs (n=4119). LSIL alone was associated with a significantly lower risk for CIN 2+ (PPV=21%) as compared with LSIL-H (PPV=40%). The results showed that the risk of CIN 2+ was intermediate for LSIL-H compared with unqualified LSIL (p<0.005) and HSIL (p<0.0001). CONCLUSIONS The current study is one of the largest LSIL-H series to date. Because of its intermediate status between LSIL and HSIL, LSIL-H should be considered a distinct diagnostic category, and specific cytomorphological criteria should be defined. The results suggest that an LSIL-H diagnostic category would aid in more rapid detection and treatment in some patients with CIN 2+.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro

STUDY QUESTION Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

Gaucher disease associated with congenital ichthyosis in the neonate

Sir: A small number of cases with Gaucher disease who are symptomatic in the neonatal period and who show ichthyotic skin changes have been described [1, 2, 5]. This association appears to be typical for neonatal Gaucher disease [4, 5]. We wish to report another case. A 2580 g female infant was born vaginally at 37 weeks gestation to a 23-yearold, gravida 3, para 1 mother who was a second degree relative of the father. Her first pregnancy ended in spontaneous abortion. Her second pregnancy resulted in the delivery of a boy who had ichthyosis, hepatosplenomegaly, cardiac defects and died 2 days after birth. The present pregnancy, labour and delivery were uneventful. The baby had a collodion skin and ectropion, eclabinm, generalized joint contractures, gross hepatosplenomegaly, jaundice, petechiae and purpura. Laboratory investigations revealed thrombocytopenia, conjugated hyperbilirubinaemia and elevated liver enzymes. The patients clinical condition deteriorated on the 3rd day and she died on the 8th day with severe septicaemia and disseminated intravascular coagulation. At postmortem examination, large macrophages, histochemically and morphologically consistent with Gancher cells were found in the liver, spleen, lymph nodes, bone marrow and thymus (Fig. 1). The parents were later tested for glucocerebrosidase activity which was 50% of normal in both. To our knowledge eight cases of neonatal Gancher disease associated with ichthyosis have been reported [1-5]. Although the reason for this association is not clear, it appears that the enzyme deficiency may be directly responsible for the skin changes in these neonates [5]. Thus, Gaucher disease should be considered in the differential diagnosis of congenital ichthyosis. In our case the diagnosis was established after the death of the second child. Recognition of this distinct Gaucher Fig. 1 A typical Gaucher cell in the lymph node parenchyma (H & E x 770)

Sentinel Lymph Node Biopsy in Pure DCIS: Is It Necessary?

Introduction. Sentinel lymph node biopsy (SLNB) in patients with pure ductal carcinoma in situ (DCIS) has been a matter of debate due to very low rate of axillary metastases. We therefore aimed to identify factors in a single institutional series to select patients who may benefit from SLNB. Material and Methods. Patients, diagnosed with pure DCIS (n = 63) between July 2000 and March 2011, were reviewed. All the sentinel lymph nodes were examined by serial sectioning (50 μm) of the entire lymph node and H&E staining, and by cytokeratin immunostaining in suspicious cases. Results. Median age was 51 (range, 30–79). Of 63 patients, 40 cases (63.5%) with pure DCIS underwent SLN, and 2 of them had a positive SLN (5%). In both 2 cases with SLN metastases, only one sentinel lymph node was involved with tumor cells. Patients who underwent SLNB were more likely to have a tumor size >30 mm or DCIS with intermediate and high nuclear grade or a mastectomy in univariate and multivariate analyses. Conclusion. In our series, we found a slightly higher rate of SLNB positivity in patients with pure DCIS than the large series reported elsewhere. This may either be due to the meticulous examination of SLNs by serial sectioning technique or due to our patient selection criteria or both.

A Rare Tumor of the Face: Malignant Nodular Hidradenoma with Dermoscopic Features Mimicking Amelanotic Melanoma

Background: Sweat gland neoplasms are rare neoplasms with less known dermoscopic features. Observations: By applying dermoscopy, we identified some common vascular features that have been defined for amelanotic melanoma such as hairpin vessels, dotted vessels, linear irregular vessels, glomerular vessels, and milky-red areas. Conclusion: Polymorphic vascular structures can also be seen in eccrine neoplasms. Further evaluations should be done to differentiate and identify the common dermoscopic features of other sweat gland tumors.

Effects of different aerobic exercise frequencies on streptozotocin-nicotinamide-induced type 2 diabetic rats: continuous versus short bouts and weekend warrior exercises

Exercise training is known to have multiple beneficial effects on type 2 diabetes mellitus (T2DM). The aim of this study was to explore the effects of aerobic exercise frequency on diabetic parameters, the histopathological structure of skeletal muscle, diabetic myopathy, and mitochondrial enzyme activity in an experimental model of T2DM.

Annular lichen planus-like keratosis: clues to pre-existing porokeratosis

Lichen planus-like keratosis (LPLK), or benign lichenoid keratosis, is usually a solitary, scaling, hyperkeratotic, papular lesion, and is generally considered as an involuting epidermal inflammatory process that leaves a pigmented lesion behind. It was first described in 1966 by Shapiro and Ackerman, since which time its clinical, histologic and even dermoscopic variants have been defined. Herein, we report an annular LPLK, displaying unusual clinical and histopathologic features.

Endogenous c-Jun N-terminal kinase (JNK) activity marks the boundary between normal and malignant granulosa cells

Granulosa cell tumor of the ovary (GCT) is a very rare tumor, accounting for only 2% of all ovarian tumors. It originates from sex cords in the ovary and can be divided into adult (95%) and juvenile (5%) types based on histologic findings. To date, no clear etiologic process has been identified other than a missense point mutation in the FOXL2 gene. Our previous works showed that c-Jun N-terminal kinase (JNK) pathway plays critical role in cell cycle progression and mitosis of normal and immortalized granulosa cells and follicle growth in rodent ovaries. These findings led us to investigate the role of JNK pathway in the granulosa cell tumor of the ovary. We used two different GCT cell lines (COV434 and KGN) and fresh GCT samples of adult and juvenile types obtained from the patients during surgery. We have discovered that endogenous kinase activity of JNK is markedly enhanced in the GCT samples and cell lines, whereas it was almost undetectable in mitotic non-malignant human granulosa cells. The inhibition of JNK pathway in GCT cell lines with two different pharmacologic inhibitors (SP600125 and AS601245) or siRNA resulted in a dose-dependent reduction in in vitro cell growth, increased apoptosis and diminished estradiol and AMH productions. JNK inhibition was also associated with a decrease in the number of cells positive for mitosis marker phospho-histone H3Ser 10 in the asynchronous cells; and diminished EdU uptake during S phase and cell cycle arrest at G2/M-phase transition in the synchronized cells. Ex vivo treatment of patient-derived GCT samples with JNK inhibitors for 24 h significantly decreased their in vitro growth and estradiol and AMH productions. Furthermore, in human GCT xenograft model, in vivo tumor growth was significantly reduced and plasma AMH levels were significantly decreased in SCID mice after administration of JNK inhibitors and siRNA. These findings suggest that targeting JNK pathway may provide therapeutic benefit in the treatment of granulosa cell tumors for which currently no curative therapy exists beyond surgery.

Analyzing esophageal squamous cell papillomas for the presence of human papilloma virus

BACKGROUND/AIMS Human Papilloma Virus (HPV) infection can be a predisposing condition for the development of squamous cell papilloma (SCP) of the esophagus, which can progress to dysplasia and to carcinoma as a result of chronic infection. The aim of the present study was to search for the presence of HPV in the esophageal SCP, and to genotype the detected HPV. MATERIALS AND METHODS Data from patients with definite diagnosis of SCP of the esophagus were identified from pathology records for two years period at different Hospitals. Slides from each patient were reviewed and samples with satisfactory papilloma tissues were submitted to molecular analysis. DNA has been isolated. DNA sequencing has been performed for genotyping HPV for all types. RESULTS Our study group consisted of 21 women and 17 men (a total of 38 patients), mean age was 41 years (range 17-67 years). Most of the papillomas were located at mid-esophagus (68%). Eight out of 38 patients (21%) had associated erosive esophagitis, and fourteen patients (36.8%) had Helicobacter Pylori (H. pylori). Of the 38 SCP analyzed, seven (19%) were positive for HPV DNA. Three of them were of genotype 6, whereas four were of genotype 16, 18, 31, 81 that are known as highly oncogenic. There were no correlations between the presence of HPV and the patients age, the presence of reflux esophagitis or H. pylori, smoking habit and the location of the papillomas. CONCLUSION The presence of high-risk type HPV in esophageal SCP may implicate a role of the virus in the pathogenesis of the esophageal tumor.