Objectives Treatment options for recurrent low grade serous ovarian carcinoma (LGSOC) are limited. Herein we describe the potential utility of next generation sequencing in identifying therapeutic targets for this rare tumour. Methods 3 patients were included in this study. DNA was extracted from ten FFPE tumour samples (5 from primary surgery and 5 from recurrences) and whole blood. Whole exomes sequencing (WES) was performed on Illumina's NextSeq platform to a depth of at least 10X. Sequence data were trimmed, aligned and single nucleotide variants and copy number alterations were called. Results All samples were microstatellite stable, 2 of the 3 patients had an elevated tumour mutational burden(TMB) (defined as >10 mutations/Mb). In the patient with low TMB we identified a class 3 'kinase-dead' BRAF variant, D594G with concordant near-whole chromosome 1 amplification, covering the NRAS proto-oncogene. Single gene testing confirmed wild type EGFR and KRAS. This lady did not respond to a trametinib. The second case identified a pathogenic NBN R43* mutation with associated non pathogenic mutations in other DNA damage response genes. This patient who had previously declined cytotoxic chemotherapy has had a partial response to platinum based chemotherapy. The third patient did not have a targetable mutation but is awaiting PD-L1 testing. Conclusions WES may be helpful in refractory LGSOC when standard treatment options have been exhausted. Two of our patients had an elevated TMB suggesting that the efficacy of immunotherapy in LGSOC should be investigated.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers ( n = 17) and lactating cows ( n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M ( n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of β-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein ( STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.

We investigated the role of the C1772T polymorphisms in exon 12 of the Hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene C1772T genotype in prostate cancer (PCa) and amplification of the hypoxic response. We identified the heterozygous germline CT genotype as an increased risk factor for clinically localised prostate cancer (Odds ratio = 6.2; p < 0.0001). While immunostaining intensity for HIF-1 alpha and VEGF was significantly enhanced in 75% of PCa specimens when compared to matched benign specimens (p < 0.0001), the CT genotype did not modulate the kinetics of HIF-1 alpha protein expression in hypoxia in vitro, and was not associated with enhanced expression of hypoxic biomarkers. This study provides the first evidence of an increased risk for clinically localised prostate cancer in men carrying the C1772T HIF-1 alpha gene polymorphism. Although our results did not suggest an association between expression of hypoxic biomarkers and genotype status, the correlation may merit further investigation.

AIMS:Alpha-methylacyl-CoA racemase (AMACR) is a sensitive and specific immunohistochemical marker of prostatic malignancy, staining 80-100% of prostatic cancers with absent staining in benign glands. However, positive staining in benign conditions as well as low rates of AMACR reactivity in prostatic cancer variants have been described. Preliminary use of AMACR immunohistochemistry in our institution has suggested lower specificity and sensitivity for prostatic cancer than initially proposed. The aim of this study was to establish true rates of AMACR reactivity in prostatic cancer and benign prostatic hyperplasia (BPH).METHODS AND RESULTS:AMACR immunohistochemistry was performed on sections from 57 prostatic cancers and 44 BPH resections. Ninety-one percent of cancers were AMACR+, with diffuse (> 75%) tumour staining in 53% of cases. Thirty-eight percent of tumours showed heterogeneous expression (1-75% tumour staining). This was significantly correlated with increased Gleason score. High-grade prostatic intraepithelial neoplasia (PIN) was AMACR+ in 87% of cancers. Eleven percent of BPH showed moderate or strong staining in benign glands, focally mimicking the malignant staining pattern.CONCLUSIONS:This study confirms heterogeneous AMACR expression in prostatic cancer and shows a correlation with Gleason score. Positive staining in BPH is also documented, thus emphasizing the importance of interpreting AMACR immunohistochemistry in the context of other findings in a diagnostic setting.

The TNM staging system represents the cornerstone for classifying patients with upper tract urothelial carcinoma (UTUC). We tested the prognostic impact of pT and pN stages on cancer-specific mortality (CSM) in a large population-based cohort of surgically treated patients with UTUC.Our analyses relied on 2299 patients treated with nephroureterectomy (NU) or segmental ureterectomy (SU) for UTUC within nine Surveillance, Epidemiology and End Results registries between 1988 and 2004. CSM rates after surgery were graphically explored using Kaplan–Meier plots. Univariable and multivariable Cox regression models tested the effect of pT and pN stages on CSM, after adjusting for tumour grade, age, gender, primary tumour location, type and year of surgery.Five years after surgery, the overall CSM-free survival rate was 77.6%. The 5-year CSM-free survival rates of pT1N0 (n = 739), pT2N0 (n = 422), pT3N0 (n = 691), pT4N0 (n = 190) and any T N1–3 (n = 257) were, respectively, 93.5 versus 86.2 versus 64.5 versus 54.7 versus 35.0%. The 5-year CSM-free survival rates of pT1–2N1–3 (n = 41) and pT3–4N1–3 (n = 216) patients were, respectively, 68.9% and 28.7% (p = 0.006). In multivariable analyses, pT and pN stages (p < 0.001), as well as tumour grade (p < 0.001), achieved independent predictor status. Advanced age adversely affected CSM-free survival (p = 0.001). Conversely, tumour location, gender, year and type of surgery did not exert independent predictor status.Durable cancer control can be expected in patients treated with NU or SU for organ-confined (pT1–2) UTUC. Conversely, the presence of non-organ-confined (pT3–4) disease and/or of nodal metastases (pN1–3) exerts a profound detrimental effect on CSM-free survival.

Promoter hypermethylation is central in deregulating gene expression in cancer. Identification of novel methylation targets in specific cancers provides a basis for their use as biomarkers of disease occurrence and progression. We developed an in silico strategy to globally identify potential targets of promoter hypermethylation in prostate cancer by screening for 5' CpG islands in 631 genes that were reported as downregulated in prostate cancer. A virtual archive of 338 potential targets of methylation was produced. One candidate, IGFBP3, was selected for investigation, along with glutathione-S-transferase pi (GSTP1), a well-known methylation target in prostate cancer. Methylation of IGFBP3 was detected by quantitative methylation-specific PCR in 49/79 primary prostate adenocarcinoma and 7/14 adjacent preinvasive high-grade prostatic intraepithelial neoplasia, but in only 5/37 benign prostatic hyperplasia (P < 0.0001) and in 0/39 histologically normal adjacent prostate tissue, which implies that methylation of IGFBP3 may be involved in the early stages of prostate cancer development. Hypermethylation of IGFBP3 was only detected in samples that also demonstrated methylation of GSTP1 and was also correlated with Gleason score > or =7 (P=0.01), indicating that it has potential as a prognostic marker. In addition, pharmacological demethylation induced strong expression of IGFBP3 in LNCaP prostate cancer cells. Our concept of a methylation candidate gene bank was successful in identifying a novel target of frequent hypermethylation in early-stage prostate cancer. Evaluation of further relevant genes could contribute towards a methylation signature of this disease.

This observational, cohort study aimed to examine the potential utility of Rapid Assessment Breast Clinics (RABC) beyond cancer detection at presentation. One thousand four hundred and twenty nine women were studied over an 18 month period. 154 (10.7%) had breast cancer – 87.7% of whom were seen expediently with 92.9% being diagnosed at one attendance. One hundred and forty three (10%) of those with a benign diagnosis were found by routine questioning to have significant familial risk separate to their reason for referral. Despite careful triage, considerable contamination of appointment allotment occurred with many who were correctly triaged as non-urgent being seen ‘urgently’. One hundred and seventy six attendees (12.3%) had neither the symptom that triggered referral, nor breast lump, nipple discharge nor family history of breast cancer, while 283 (19.8%) had no objective clinical or radiological abnormality. Although RABC reliably categorise malignant versus non-malignant diagnoses despite cluttering by low risk women, a significant proportion of non-cancer patients still require address of future risk rather than reassurance of their present status alone.

he assessment of cervical lymph node metastases in patients with squamous cell carcinoma of the head and neck is essential for the clinical management of the disease. Staging of the neck, which constitutes a diagnostic problem because of the large number of lymph nodes located in this area (1), determines the extent of treatment modalities such as surgery or radiotherapy. In addition to palpation, staging of the neck increasingly relies on imaging modalities such as CT and MRI that use lymph node size and central lucency as criteria for detection of metastatic disease (2—4). Although lymph nodes 10 mm in diameter are generally considered tobepositive(5), lymphnodeenlargement mayalsobethe result of reactive and inflammatory processes that may limit the accuracy of CT and MRJ. Moreover, detection of micro metastases in small nodes still remains a difficult issue (6,7). To overcome these limitations, ultrasound-guided fine needle aspiration cytology (USG-FNAC) was introduced in recent years. By combining detection by sonography with tissue characterization by microscopy (8,9), this technique has reached high accuracy levels in the diagnosis of regional spread in clinically negative necks and necks with lymphad enopathies. However, the accuracy of USG-FNAC depends on the skill of the sonographer and the cytopathologist, and the technique requires multiple aspirations. As an alternative modality, PET has been used for the staging of the neck. 18F-fluorodeoxyglucose (FDG) is able to detect metastases measuring 4 mm or more, but the relatively high false positive rate caused by uptake in reactive lymph nodes (10) has led to the use of other compounds, such as L-[―CH3]methionine (11) and L-1-[―C]-tyrosine (12,13), that appear to be more accurate in differentiating cancerous from inflammatory cells. The value of SPECTwith @°1TI chloride, in combinationwith MRI (particularly short inversion-time inversion recovery [STIRI Se quences that suppress fat signals) to detect and characterize cervicallymphadenopathies (nodes @ 1cm),andexvivolymph node @°1Tl uptakewerestudiedin patientswithsquamouscell carcinomaofthe headand neck.Methods: PreoperativeSPECT and MRI, displayed in similar planes, were compared with the histologicfindingsin 15 neck dissectionspecimensfrom 12 patients with squamous cell carcinoma of the head and neck (9 with unilateraland 3 with bilateralneckdissection).Resultswere evaluatedtopographicallywith regardtothe lymphnodecompart ments (levels)of the neck. In addition, in 8 of these patients,the

AIM:To investigate and compare topoisomerase II-alpha expression in benign prostatic hyperplasia (BPH), prostate cancer of varying Gleason scores and hormone-insensitive prostate cancer.METHODS:The immunohistochemical expression of topoisomerase II-alpha antibody in the above-mentioned diagnostic categories was investigated and compared.RESULTS:Increased expression of topoisomerase II-alpha was seen in the prostate cancers of Gleason scores 7 and 8-10 (p = 0.000) compared with prostate cancers of Gleason score 6 and BPH (p = 0.245). Statistically significant differences were found in the topoisomerase II-alpha gene expression between prostate cancers categorised by Gleason Score. Also, increased expression of topoisomerase II-alpha was seen in the known hormone-resistant prostate carcinomas compared with prostate cancers with no hormone treatment in the subgroup with Gleason scores 8-10, which approached statistical significance (p = 0.081). No statistically significant difference was observed in topoisomerase II-alpha expression between the groups with BPH and prostate carcinoma of Gleason score 6 (p = 0.245).CONCLUSION:Topoisomerase II-alpha expression was found to increase with the known prognostic marker Gleason score and with hormone insensitivity. Objective evidence is provided for clinical trials with drugs targeting topoisomerase II-alpha to be targeted to patients with prostate cancers of Gleason Score >6 and, in particular, prostate cancers of Gleason Scores 8-10.