Association between pelvic inflammatory disease, infertility, ectopic pregnancy and the development of ovarian serous borderline tumor, mucinous borderline tumor and low-grade serous carcinoma.

OBJECTIVE: Risk factors for ovarian borderline tumors and low-grade serous carcinoma (LGSC) are poorly understood. The aim of this study was to examine the association between infertility, pelvic inflammatory disease (PID), endometriosis, ectopic pregnancy, hysterectomy, tubal ligation and parity and the risk of serous borderline tumor (SBT), mucinous borderline tumor (MBT) and LGSC. METHODS: This was a population-based cohort study using linked administrative and hospital data. Participants were 441,382 women born between 1945 and 1975 who had been admitted to hospital in Western Australia between 1 January 1980 and 30 June 2014. We used Cox regression to estimate hazard ratios (HRs). RESULTS: We observed an increased rate of SBT associated with infertility, PID and ectopic pregnancy (HRs and 95% CIs were, respectively, 1.98 (1.20-3.26); 1.95 (1.22-3.10) and 2.44 (1.20-4.96)). We did not detect an association between any of the factors under study and the rate of MBT. A diagnosis of PID was associated with an increased rate of LGSC (HR 2.90, 95% CI 1.21-6.94). CONCLUSIONS: The association with PID supports the hypothesis that inflammatory processes within the upper gynaecological tract and/or peritoneum may predispose to the development of SBT and LGSC.

Novel BRAF and KRAS Mutations in Papillary Thyroid Carcinoma Arising in Struma Ovarii.

Papillary carcinomas of thyroid type rarely arise within struma ovarii. There are limited data on the immunohistochemical and molecular features of these tumors. Three cases of papillary carcinoma arising in struma ovarii (PCSO) were identified. The clinicopathological features were reviewed and immunohistochemical staining for HBME-1, cytokeratin (CK) 19, and CD56 was performed. Tumor DNA was sequenced for somatic mutations using a panel of 26 oncogenes, with a particular focus on BRAF and KRAS mutations. The patients were aged 22, 48, and 55 years. All cases were FIGO stage IA. Two tumors were of classical histological type, and one was a follicular variant papillary carcinoma. All tumors expressed HBME-1 and two were positive for CK19. CD56 was negative in all three cases. One tumor demonstrated a BRAF G469A mutation in exon 11, and in a second case, a KRAS Q61K double base mutation in exon 3 was detected. These mutations have not been described previously in PCSO. No mutations were detected in the benign follicular components of the tumors adjacent to the malignant papillary tissue. None of the patients had tumor recurrence on clinical follow-up (range 11 months to 8½ years). HBME-1, CK19, and CD56 are useful immunohistochemical markers of PCSO. Novel BRAF and KRAS mutations were identified in two of three tumors suggesting that mutations in PCSO may differ from those commonly identified in papillary carcinoma of the eutopic thyroid. The clinical significance of these mutations is uncertain but follow-up data in this small series support the generally good prognosis of PCSO.

Comparison of cold knife cone biopsy and loop electrosurgical excision procedure in the management of cervical adenocarcinoma in situ: What is the gold standard?

OBJECTIVE: To compare the outcomes of patients with cervical adenocarcinoma in situ (ACIS) treated with cold knife cone (CKC) biopsy or loop electrosurgical excision procedure (LEEP) for the treatment of cervical adenocarcinoma in situ (ACIS). STUDY DESIGN: This is a retrospective, population-based cohort study of Western Australian patients with ACIS diagnosed between 2001 and 2012. Outcomes included pathological margin status and the incidence of persistent or recurrent endocervical neoplasia (ACIS and adenocarcinoma) during follow-up (<12 months) and surveillance (≥12 months) periods. RESULTS: The study group comprised 338 patients including 107 (32%) treated initially by LEEP and 231 (68%) treated by CKC biopsy. The mean age was 33.2 years (range 18 to 76 years) and median follow-up interval was 3.6 years (range <1 year to 11.8 years). Overall, 27 (8.0%) patients had ACIS persistence/recurrence while 9 (2.7%) were diagnosed with adenocarcinoma during the follow-up and surveillance periods. No patient died of cervical cancer within the study period. There were no significant differences in the incidence of persistent and/or recurrent endocervical neoplasia according to the type of excisional procedure. Patients with positive biopsy margins were 3.4 times more likely to have disease persistence or recurrence. CONCLUSION(S): LEEP and CKC biopsy appear equally effective in the treatment of ACIS for women wishing to preserve fertility. Patients undergoing conservative management for ACIS should be closely monitored, particularly if biopsy margins are positive in initial excision specimens. Patients and their clinicians should be aware of the potential risks of residual and recurrent disease.

NUT protein immunoreactivity in ovarian germ cell tumours.

The aim of this study was to investigate NUT (nuclear protein in the testis) expression in ovarian germ cell tumours (GCTs). Immunostaining for NUT protein was performed in 10 mature cystic teratomas and in 49 malignant ovarian GCTs including 15 pure dysgerminomas, six dysgerminomas associated with gonadoblastoma, nine yolk sac tumours, 12 immature teratomas, and seven mixed malignant tumours. Only nuclear staining was considered a positive finding although cytoplasmic staining was noted when present. Thirty-seven (76%) malignant GCTs were NUT positive but staining was usually of weak to moderate intensity and observed in a relatively small proportion of neoplastic cells. Staining in immature teratomas and yolk sac tumours was restricted to foci of hepatoid and intestinal/glandular differentiation, where both nuclear and cytoplasmic reactivity were observed. In dysgerminoma associated with gonadoblastoma only the in situ and invasive germ cell elements were NUT positive. Nuclear staining was not seen in benign teratomas. Most malignant ovarian GCTs express NUT protein, albeit focally, and this should be considered when evaluating immunostaining in the differential diagnosis of poorly differentiated malignancies, particularly NUT midline carcinoma. Since NUT protein appears to play a role in normal germ cell maturation it may influence intestinal or hepatoid differentiation within malignant GCTs.

Fascin expression in cervical normal squamous epithelium, cervical intraepithelial neoplasia, and superficially invasive (stage IA1) squamous carcinoma of the cervix.

The aims of this study were to: investigate fascin expression in normal cervix, cervical intraepithelial neoplasia (CIN), and stage IA1 squamous cell carcinoma (SCC).Fascin immunostaining was performed in cervical biopsies showing normal squamous epithelium (n=10), CIN 1 (n=10), CIN 2-3 without invasion (n=11), and CIN 2-3 adjacent to SCC (n=40); SCC was also present in 27 of the latter cases.Fascin expression in normal squamous epithelium was restricted to basal and parabasal cells, whereas there was increased staining in immature squamous metaplasia and in most CIN lesions. Full thickness staining was more frequent in high grade CIN adjacent to invasion than in CIN 2-3 alone. Eighteen SCCs (67%) were fascin positive and seven cases showed accentuated staining at the tumour-stromal interface (invasive front). There was no consistent relationship between fascin expression in CIN lesions and in corresponding carcinomas. Fascin staining in reactive stromal cells sometimes made identification of the invasive neoplastic cells difficult.Fascin is overexpressed in most CIN lesions and this may be a marker of increased invasive potential in high grade CIN. However, fascin staining does not distinguish low and high grade CIN or in situ and invasive squamous neoplasia. Therefore fascin has limited diagnostic utility in demonstrating superficial stromal invasion.

Fascin and cyclin D1 immunoreactivity in non-neoplastic vulvar squamous epithelium, vulvar intraepithelial neoplasia and invasive squamous carcinoma: correlation with Ki67 and p16 protein expression.

AIMS: To investigate cyclin D1 and fascin immunoreactivity in normal, reactive and neoplastic vulvar skin correlating the findings with p16 protein and Ki67 expression. METHODS: 66 vulvar biopsy or resection specimens demonstrating normal appearances, reactive epidermal changes, usual-type vulvar intraepithelial neoplasia (uVIN), differentiated-type VIN (dVIN), p16-positive squamous cell carcinoma (SCC) and p16-negative SCC were examined immunohistochemically for cyclin D1, fascin, Ki67 and p16 protein. Where applicable, expression patterns were compared in microanatomically distinct areas, particularly at the invasive front (deep tumour margin) of SCC. RESULTS: Normal epidermis showed parabasal Ki67 and cyclin D1 staining while fascin labelled cells in the lower one-third of the epithelium. Reactive and dVIN specimens demonstrated mildly increased Ki67 and cyclin D1 expression that maintained parabasal polarity, whereas uVIN and p16-positive SCC were characterised by loss of cyclin D1 staining. However, in 14 of 20 p16-positive SCC small infiltrative tumour groups and single infiltrating cells at the invasive front showed a cyclin D1-positive/ Ki67-negative phenotype. In contrast, p16-negative SCC generally showed diffuse and concordant cyclin D1 and Ki67 labelling, including at the invasive margin. Fascin expression was increased in all VIN and SCC lesions. CONCLUSIONS: Variations in cyclin D1 and Ki67 expression between p16-positive and p16-negative vulvar SCCs suggest different mechanisms of invasion in these tumour subgroups. Fascin is upregulated in vulvar squamous neoplasia but immunostaining does not discriminate in situ from invasive lesions nor putative human papilloma virus (HPV)-associated and HPV-independent SCCs.

DICER1 hotspot mutations in non-epithelial gonadal tumours.

BACKGROUND: Non-epithelial gonadal tumours largely comprise sex cord-stromal tumours (SCSTs) and germ cell tumours (GCTs). Specific somatic mutations in DICER1, a microRNA maturation pathway gene, have been identified in these tumours. We conducted a study that aimed to confirm, refine and extend the previous observations. METHODS: We used Sanger sequencing to sequence the RNase IIIa and IIIb domains of DICER1 in 154 gonadal tumours from 135 females and 19 males, as well as 43 extra-gonadal GCTs from 26 females and 17 males. RESULTS: We identified heterozygous non-synonymous mutations in the RNase IIIb domain of DICER1 in 14/197 non-epithelial tumours (7.1%). Mutations were found in 9/28 SCSTs (32%), 5/118 gonadal GCTs (4.2%), 0/43 extra-gonadal GCTs and 0/8 miscellaneous tumours. The 14 mutations affected only five residues: E1705, D1709, E1788, D1810 and E1813. In all five patients where matched and constitutional DNA was available, the mutations were only somatic. There were no mutations found in the RNase IIIa domain. CONCLUSION: More than half (8/15) of Sertoli-Leydig cell tumours (SLCTs) harbour DICER1 mutations in the RNase IIIb domain, while mutations are rarely found in GCTs. Genetic alterations in SLCTs may aid in classification and provide new approaches to therapy.

Transtubal spread of endometrial carcinoma: correlation of intra-luminal tumour cells with tumour grade, peritoneal fluid cytology, and extra-uterine metastasis.

AIMS: To assess the significance of intra-luminal tumour cells (ILTC) within the fallopian tubes of patients with endometrial carcinoma, with emphasis on high grade histological subtypes. METHODS: Complete histological examination of fallopian tube tissue was performed in 36 low grade endometrioid adenocarcinomas and in a consecutive series of 226 high grade endometrial malignancies including 92 serous carcinomas, 64 grade 3 endometrioid carcinomas, 26 carcinosarcomas, 25 undifferentiated/dedifferentiated carcinomas, and 19 clear cell carcinomas. The presence of ILTC was correlated with peritoneal fluid cytology, histologically confirmed peritoneal tumour spread, and lymph node metastases. RESULTS: ILTC were identified in 26% and 3% of high and low grade carcinomas, respectively. The presence of ILTC correlated strongly with positive peritoneal fluid cytology and with peritoneal metastasis in high grade tumours (both p < 0.001), and there was also a correlation with lymph node metastasis (p = 0.049). ILTC were more common in serous and undifferentiated carcinomas (>30%) but the differences between the high grade tumour subtypes were not statistically significant. CONCLUSIONS: These findings suggest that ILTC associated with high grade endometrial cancers have the capacity to implant and invade the peritoneal cavity. Positive cytology in such cases may be more significant than in low grade tumours. Complete tubal examination may provide additional prognostic information in high grade endometrial carcinoma.

Immunohistochemical expression and prognostic significance of oestrogen receptor-alpha, oestrogen receptor-beta, and progesterone receptor in stage 1 adult-type granulosa cell tumour of the ovary.

AIMS: To assess oestrogen receptor (ER)α, ERß, and progesterone receptor (PR) expression in stage I ovarian adult-type granulosa cell tumours (AGCTs) and correlate the findings with clinical outcome. METHODS: ERα, ERß and PR immunohistochemistry was performed on 56 primary, stage I AGCTs. Twelve cases (21%) recurred and hormone receptor staining was compared in the corresponding primary and metastatic tumours. RESULTS: All primary AGCTs expressed ERß and PR, usually with strong and diffuse staining, whereas only 20% of tumours were focally ERα positive. There was no correlation between ERα or PR expression and outcome. However, primary AGCTs with low ERß expression had a significantly higher risk of recurrence. In contrast, all metastatic tumours exhibited strong ERß staining. No relationship between ER staining and tumour morphology was identified but there was more consistent PR expression in cells at the tumour-stromal interface. CONCLUSIONS: Primary AGCTs typically show an ERα negative and ERß/PR positive immunophenotype. Low ERß expression is an adverse prognostic factor in primary AGCT but metastatic tumours often show up-regulation of ERß. Local microenvironmental factors may influence PR expression. Hormone receptor expression in AGCT may become increasingly relevant due to developments in selective therapy.

CD147 (EMMPRIN) and matrix metalloproteinase-2 expression in uterine endometrioid adenocarcinoma.

Low-grade uterine endometrioid adenocarcinomas (EACs) may exhibit a distinct pattern of myometrial invasion characterized by the presence of microcystic, elongated, and fragmented ('MELF') glands but the factors influencing this pattern of invasion are not known. Immunohistological expression of CD147 (extracellular inducer of matrix metalloproteinase, EMMPRIN) and matrix metalloproteinase-2 (MMP2) was studied in 22 EAC and the results compared between the 'conventional' neoplastic glands, foci of MELF-type invasion, and in the tumor-associated stroma. The conventional tumor areas showed strong membranous expression of CD147, and staining was more consistently present toward the central (luminal) aspect of the larger glands. In contrast, the tumor cells showed only focal expression of MMP2, and in some cases CD147 and MMP2 staining was inversely correlated. The neoplastic epithelium within MELF areas usually was negative for both CD147 and MMP2. However, there was consistent MMP2 expression in the reactive stroma that characteristically surrounds foci of MELF pattern invasion. This micro-anatomical variation in protein expression within EAC suggests a functional alteration in the neoplastic epithelium during the invasive process. It is likely that stromal MMP2 plays a role in potentiating invasion in these tumors but this may be regulated by factors other than CD147.

KRAS mutation and microsatellite instability in endometrial adenocarcinomas showing MELF-type myometrial invasion.

BACKGROUND: Some uterine endometrioid adenocarcinomas exhibit a distinctive morphological phenotype characterised by the formation of microcystic, elongated and fragmented (MELF) glands. Immunohistochemical studies have suggested that MELF-type changes represent an epithelial-mesenchymal transition which has been associated with KRAS activation in various tumours. AIMS: To investigate the molecular characteristics of endometrial tumours showing MELF, with particular reference to the frequencies of KRAS and BRAF mutations and of microsatellite instability (MSI). METHODS: MSI, and KRAS and BRAF mutation status, were assessed in 33 low-grade endometrial adenocarcinomas showing MELF features and the results compared with 33 control cases exhibiting a 'conventional' pattern of myometrial invasion. Standard histological parameters were also reviewed. RESULTS: Tumours with a MELF pattern of myometrial invasion showed more frequent vascular invasion and focal mucinous differentiation. KRAS mutations were more frequent in MELF positive than MELF negative tumours (45% vs 30%), but this difference was not statistically significant. BRAF mutations were not identified in any of the cases. MSI was identified in 20% of cases overall but did not correlate with the MELF phenotype. CONCLUSIONS: Mutations in KRAS and BRAF genes are not directly implicated in the development of a MELF pattern of invasion in endometrial carcinoma. However, RAS-associated signalling pathways could be activated through other genetic or epigenetic mechanisms. The characterisation of such alterations may become increasingly important as novel therapies are developed that target mediators involved in tumour invasion.

Diagnostic value and implications of vimentin expression in normal, reactive and neoplastic endocervical epithelium.

AIMS: To assess vimentin immunoreactivity in normal, reactive and neoplastic endocervical epithelium, and compare the results with p16 protein, Ki-67 and bcl-2 expression. METHODS: Sixty-two cervical biopsy specimens including normal endocervical epithelium, tubo-endometrioid metaplasia, adenocarcinoma in situ, stratified mucin producing intraepithelial lesions (SMILE), and invasive adenocarcinomas were stained immunohistochemically for vimentin and for p16 protein, Ki-67 and bcl-2. Twelve cases also included areas of high grade cervical intraepithelial neoplasia (CIN). RESULTS: Normal endocervical epithelium usually showed subtle but distinct sub-nuclear and delicate lateral cell border vimentin expression while tubo-endometrioid metaplasia exhibited more diffuse cytoplasmic immunoreactivity. Usually adenocarcinoma in situ was completely negative and therefore vimentin staining sharply distinguished the benign and neoplastic epithelial elements. The SMILE lesions and high grade CIN were also vimentin negative in most cases. Most invasive adenocarcinomas were not stained but focal vimentin immunoreactivity was observed in 7/18 cases, and was restricted to small glands and infiltrating cell clusters at the deep (advancing) tumour margin. CONCLUSIONS: Normal endocervical cells often exhibit vimentin staining, and this is increased in reactive and metaplastic situations, whereas adenocarcinoma in situ is usually completely negative. Therefore vimentin is a useful additional diagnostic marker in the assessment of problematic cervical glandular lesions. The localised re-expression of vimentin at the deep margin of some endocervical adenocarcinomas may be relevant to the process of tumour progression and invasion in these cases.

MELF pattern invasion in endometrial carcinoma: association with low grade, myoinvasive endometrioid tumours, focal mucinous differentiation and vascular invasion.

AIMS: To investigate the frequency of microcystic, elongated and fragmented (MELF) pattern invasion in endometrial carcinoma and its association with other pathological findings. METHODS: The histology slides from 170 consecutively accessioned endometrial malignancies were reviewed to identify cases showing MELF type invasion. Histological subtype, tumour grade, presence of squamous and mucinous differentiation, and frequency of lymphovascular space invasion (LVSI) were compared in MELF positive and negative cases. Additional stains for cytokeratin and stromal mucin were performed in the MELF positive tumours and immunohistochemistry to demonstrate vascular endothelium was performed in 12 selected cases. RESULTS: There were 133 endometrioid adenocarcinomas (EEC) and 37 non-endometrioid carcinomas. MELF type invasion was identified in 27 cases (15.9%) all of which were FIGO grade 1 or 2 myoinvasive adenocarcinomas of endometrioid type. The histochemical and immunohistochemical stains were useful in demonstrating the relatively subtle invasive foci in some tumours. MELF positive cases more frequently showed focal mucinous differentiation and LVSI. CONCLUSIONS: MELF type invasion appears restricted to low grade, myoinvasive carcinomas of endometrioid type and is more common in tumours exhibiting focal mucinous differentiation. These associations suggest that MELF changes represent a specific tumour-stromal interaction rather than a degenerative process. Tumours exhibiting MELF pattern invasion should be carefully examined for LVSI. Ancillary techniques, in particular cytokeratin immunohistochemistry, are useful in diagnostic assessment.

Acquired lymphangiectasia ('lymphangioma circumscriptum') of the vulva: a report of eight cases.

AIMS: To present the clinico-pathological findings of eight cases of acquired vulval lymphangiectasia (AVL) with discussion of the terminology and differential diagnosis. METHODS: Vulvectomy or biopsy specimens from eight patients with AVL were reviewed. All patients had undergone surgery, lymphadenectomy and/or radiotherapy, most commonly for carcinoma of the cervix, up to 26 years prior to presentation with the lymphangiectatic lesions. Immunohistochemistry for CD31, CD34, D2-40, p53 and p16 was performed in each case. RESULTS: The original clinical and pathological diagnoses were most frequently 'lymphangioma circumscriptum' but viral infection was considered in some cases. All specimens showed dermal lymphangiectasia associated with marked reactive epidermal hyperplasia. The lymphatic endothelial cells showed CD31 and D2-40 expression but CD34 was negative. The keratinocytes showed focal p53 immunoreactivity in four cases. CONCLUSIONS: AVL is the preferred nomenclature for the lesions presented herein. The clinical and histological features usually are characteristic but the differential diagnosis may include condyloma and differentiated type vulval intraepithelial neoplasia (VIN). Immunohistochemistry may be helpful but lack of CD34 expression should be noted and may prove useful in the differential diagnosis of other vulval vascular lesions. Focal p53 protein immunoreactivity should not be considered indicative of differentiated type VIN in this clinical setting.

WT1 expression in endometrioid ovarian carcinoma with and without associated endometriosis.

AIMS: To determine how frequently endometrioid ovarian carcinomas (EOC) express WT1 protein, and to correlate the results with the presence of endometriosis and p53 immunoreactivity. METHODS: Forty-one grade 1-2 EOC were stained immunohistochemically for WT1 and p53 proteins. Twenty-one tumours were associated with endometriosis and 20 cases lacked such an association. WT1 expression in the tumour cell nuclei and/or cytoplasm was recorded. Nuclear p53 staining was assessed as diffuse (>50% cells positive), focal, or negative. RESULTS: Four of the 20 (20%) tumours in the endometriosis negative group showed nuclear WT1 staining, while none of the endometriosis-associated EOC was positive (p < 0.05). Two of the immunoreactive cases exhibited sertoliform/sex cord-like patterns. Focal cytoplasmic WT1 labelling was present in seven EOC, three of which showed sertoliform, spindle cell or corded and hyaline patterns. There was no correlation between WT1 expression and p53 immunoreactivity. CONCLUSIONS: Nuclear WT1 expression is present in a minority of EOC and this should be considered if immunohistochemistry is used as an adjunct in sub-typing ovarian carcinomas. The negative correlation of WT1 staining with endometriosis supports the possibility that some EOC, including unusual histological variants, arise from the ovarian surface epithelium. Further studies of EOC should document any association with endometriosis.