Reducing the radicality of surgery for vulvar cancer: are smaller margins safer?

INTRODUCTION: Vulvar cancer accounts for ~4% of all gynecological malignancies and the majority of tumors (>90%) are squamous cell (keratinizing, ~60% and warty/basaloid, ~30%). Surgical excision forms the foundation of treatment, with resection margin status being the single most influential factor when predicting clinical outcome. There has been a paradigm shift concerning surgical approaches and radicality when managing vulvar cancer within recent times, largely owing to a desire to preserve vulvar structure and function without compromising oncological outcome. As such the safety of the size of resection margin has been called into question. In this narrative review we consider the current literature on the safety of resection margins for vulvar cancer. EVIDENCE ACQUISITION: PubMed, Medline and the Cochrane Database were searched for original peer-reviewed primary and review articles, from January 2005 to January 2020. The following search terms were used vulvar cancer surgery, vulvar squamous cell carcinoma, excision margins, adjuvant radiation. EVIDENCE SYNTHESIS: A pathological tumor margin of <8 mm has been widely considered to indicate "close" margins. This measurement after fixation of the tumor is considered comparable to a surgical resection margin of around 1cm, following an estimated 20% tissue shrinkage after formalin fixation and a 1-2cm clinical surgical margin in order to achieve the 8 mm final pathological margin. CONCLUSIONS: A surgical resection margin of 2-3mm does not appear to be associated with a higher rate of local recurrence than the widely used limit of 8 mm. As such the traditional practice of re-excision or adjuvant radiotherapy based on "close" surgical margins alone needs to be closely evaluated, since the attendant morbidity associated with these procedures may not be outweighed by oncological benefit.

The V-Y gluteal fold advancement flap: Outcomes following radical surgery for vulvar malignancies.

OBJECTIVE: To evaluate the feasibility and complication rate of the V-Y gluteal fold flap in surgery for vulvar cancer. METHODS: From June 2015 to June 2018, 62 patients surgically treated for vulvar cancer were included in the study. Twenty-three (37.1%) underwent plastic reconstructive surgery with V-Y advancement flaps. RESULTS: The mean surgical time was longer for patients undergoing V-Y flap surgery. The margins were positive in six patients (9.7%), close (<8 mm) in 10 (16.1%), and adequate (>8 mm) in 46 (74.2%). Six (9.7%) patients had dehiscence and two (3.2%) patients suffered from necrosis. In patients undergoing V-Y flap reconstruction, two (8.7%) had a wound dehiscence, no patients had necrosis. In patients undergoing direct closure, four (10.3%) had wound dehiscence and two (5.1%) had necrosis. CONCLUSIONS: V-Y gluteal fold advancement technique is a safe procedure, performed in a single surgical session with minimal increase in surgical time and low wound healing complications. Use of this technique was correlated with an increased rate of adequate surgical margins (<8 mm) and reduced need for adjuvant radiotherapy.

Predictors of postoperative overall and severe complications after surgical treatment for endometrial cancer: The role of the fragility index.

OBJECTIVE: To evaluate the predictive value of obesity, comorbidities, and fragility on overall and severe complication rate and survival among patients surgically treated for endometrial cancer. METHODS: Consecutive patients with endometrial cancer treated at the Royal Infirmary Hospital of Edinburgh from June 1, 2015, to June 30, 2017, were retrospectively enrolled in an observational study. Considering pre-existing medical conditions, comorbidities, and complications, modified fragility index (mFI) was calculated. Logistic regression was used to evaluate predicting variables of overall (G1-G4) and severe (G3-G4) complication rate. RESULTS: One hundred patients were surgically treated for endometrial cancer. Elevated mFI >3 was related to a statistically higher access rate to the high dependency unit (HDU) or intensive care unit (ITU) (33.3% vs 6.6%, P=0.013). Overall, 31 women had postoperative complications. Using multivariate analysis, it was shown that undergoing laparotomy (odds ratio [OR] 7.06, 95% confidence interval [CI] 2.52-19.71; P<0.001) and having an mFI >3 (OR 7.19, 95% CI 1.43-36.25; P=0.021) were independent predictors of overall complications (G1-G4). Moreover, only smoking (OR 5.01, 95% CI 1.15-21.75; P=0.031) and mFI >3 (OR 5.16, 95% CI 1.07-24.94; P=0.047) were independent factors for severe complications (G3-G4). CONCLUSION: Modified fragility index was an important predictor of complications among patients treated for endometrial cancer and could be a useful tool for assisting clinicians in perioperative management.

Secreted and intracellular cytokines are complementary measures for human monocytes treated with Toll-like receptor agonists.

Cytokine production by human peripheral blood mononuclear cells including monocytes, is frequently assessed by measuring secreted cytokines using enzyme linked immunosorbent assay (ELISA), whereby the total concentration of one cytokine of interest is obtained without information regarding the cell type responsible for making the cytokine. Cytokines can be retained inside the cell using protein transport inhibitors. Subsequent analysis by flow cytometry not only identifies the cell type producing the cytokine but can semi-quantitate the amount of cytokine produced by measuring the geometric mean fluorescence intensity (gMFI) and is amenable to analyzing more than one protein associated with the same cell (multiplexing). We hypothesized that a more comprehensive and biologically meaningful cytokine profile could be acquired by measuring both secreted and the retained intracellular cytokines in parallel cultures of magnetic-sorted CD14+ monocytes. Peripheral monocytes were isolated from 18 healthy donors and treated with standardized molecules that stimulate cytokine production; Toll-like receptor (TLR)4 agonist (lipopolysaccharide, LPS) or TLR7/8 agonist (R848). Pro-inflammatory cytokines (interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)) secreted into the culture medium were measured by ELISA. Parallel cultures were treated with LPS and R848 in the presence of brefeldin A (protein transport inhibitor) and the accumulated intracellular cytokines measured by flow cytometry. Each cytokine (IL-6/IL-8/TNF) gave a unique general pattern when secreted versus intracellular cytokine measurements (frequency and gMFI) were plotted to determine correlation. For monocytes treated with the TLR4 agonist, secreted IL-8 correlated with the frequency of IL-8 positive cells (R = 0.559, p = .016) and not with the amount (gMFI) of IL-8 per cell. In contrast, monocytes treated with the TLR7/8 agonist showed no correlation of secreted IL-8 with the frequency of IL-8 positive cells, but with this treatment secreted IL-6 was correlated with an increase in the frequency of IL-6 positive cells (R = 0.501, p = .034). TNF secretion from monocytes treated with either the TLR4 or TLR7/8 agonist did not correlate with the frequency or gMFI of TNF positive cells. However, there were significant correlations between the TLR4 and TLR7/8 induced TNF response (secreted and gMFI). We conclude that there are fundamental differences in secreted and intracellular IL-6/IL-8/TNF production after monocytes are treated with TLR agonists. Furthermore, secreted and intracellular cytokine analyses are complementary measures that should be used in parallel to explore inflammatory response and cytokine biology.

Borderline Ovarian Tumors: Fifteen Years' Experience at a Scottish Tertiary Cancer Center.

OBJECTIVES: Since the recognition of borderline ovarian tumors (BOTs) in the 1970s, the management of this subset of epithelial ovarian tumors has presented a challenge to clinicians. The majority present at an early stage, but their diagnosis is often only made following surgery, hence the heterogeneity of surgical management. Borderline ovarian tumors are morphologically diverse, and their behavior is subsequently also heterogeneous. We aimed to assess recurrence rates and the rate of malignant transformation in patients diagnosed with BOT. Secondary objectives included a review of current management and assessment of tumor markers, stage, cyst dimensions, and the presence of micropapillary features as prognostic indicators of recurrence. METHODS: This retrospective cohort study included all patients treated with BOT between 2000 and 2015 in the southeast region of Scotland. Clinical, surgicopathological, and follow-up data were collated. Data were analyzed with reference to recurrence and malignant transformation. RESULTS: Two hundred seventy-five patients underwent treatment for BOT in the study period. Surgical management was highly variable. A diagnosis of recurrent/persistent BOT or ovarian malignancy following initial treatment of BOT was rare, with only 12 (4%) of 275 cases. There were 7 cases (3%) of ovarian malignancy. Advanced International Federation of Gynecology and Obstetrics stage was the most prominent prognostic factor. Elevated preoperative serum CA-125 and the presence of micropapillary features correlated with advanced stage at presentation. With a lack of clear guidance, follow-up was highly variable with a median of 43 months (0-136 months). CONCLUSIONS: To our knowledge, this study is the largest BOT cohort in the United Kingdom. Recurrent disease is rare in optimally staged, completely resected, early-stage BOT, without high-risk features. Caution is needed in women electing not to undergo completion staging after diagnosis and in those opting for a fertility-preserving approach. Thorough informed consent and clear plans for surveillance and follow-up are needed with consideration of delayed completion surgery as appropriate.

Performance of the linear array HPV genotyping test on paired cytological and formalin-fixed, paraffin-embedded cervical samples.

Detection and genotyping of human papillomavirus (HPV) from formalin-fixed, paraffin-embedded (FFPE) samples may be difficult when using assays based on amplification of large fragments. The objective of the present study was to investigate the performance of the Linear Array HPV Genotyping Test (Linear Array) on FFPE cervical cone biopsy specimens using paired cytologic samples obtained immediately before the conization as a criterion standard. Thirty-nine samples of grade 2 or higher cervical intraepithelial neoplasia were selected; all of the corresponding cytological samples were positive by the Linear Array and had a report of atypical squamous cells of undetermined significance or worse. A valid Linear Array test result was obtained for 38 FFPE specimens (97.4%, 95% CI 88.0 to 99.9). Specifically, 34 were HPV-positive (89.5%, 95% CI 76.5 to 96.9) and 4 were HPV-negative (10.5%, 95% CI 3.4 to 23.5). The overall agreement of the results obtained for the cytologic and histologic paired samples was good (Cohen's κ = 0.85, SE = 0.082, P = 0.000). Further analysis of samples with negative or invalid Linear Array test results, both modifying the nucleic acids extraction protocol and using the INNO-LiPA assay, suggested that failure of the Linear Array test in HPV detection from tissues was probably due to DNA fragmentation. Parallel analysis of paired FFPE and cytologic samples is extremely useful for evaluation of the efficiency of PCR-based assays in HPV detection and genotyping from tissue samples. In the present study, false-negative results were obtained in a limited percentage of cases, our data depicting the successful performance of the Linear Array test on FFPE samples.

No increase in the expression of key unfolded protein response genes in HCV genotype 3 patients with severe steatosis.

Although hepatic steatosis is common in patients infected with HCV, the mechanisms leading to cellular triglyceride retention are obscure. A role for the Unfolded Protein Response (UPR) has been postulated, either through its activation or dysfunction. In this study we set out to investigate the expression of key UPR genes in HCV genotype 3 patients with moderate to severe steatosis. RNA was extracted from liver obtained by percutaneous biopsy and key genes from the UPR were semi quantified using real-time PCR. We compared values in patients with minimal steatosis to those with high steatosis. Patients with high steatosis were younger (44.6 ± 2.4 vs. 37.4 ± 2.1, p<0.05) and had higher hepatic viral RNA loads (1.00 ± 0.21 vs. 3.98 ± 0.22, p<0.05). We found no significant difference in the expression of UPR genes, except for a small increase in EDEM1 in the high steatosis group (1.00 ± 0.13 vs. 1.38 ± 0.09, p<0.05). In conclusion, despite a four-fold greater concentration of HCV RNA in tissue with a high level of steatosis, we found no change in the expression of key UPR related genes, except for a only a modest up-regulation of EDEM1. Our data does not support a sustained change in expression of UPR genes in the steatogenesis of HCVGT3 infected human liver.

Expression of common housekeeping genes is affected by disease in human hepatitis C virus-infected liver.

BACKGROUND: Comparative gene expression is commonly determined with reference to the expression of a housekeeping gene (HKG), the level of which is assumed to be unregulated. There are little data to date on the effect of disease on the expression of classic HKGs in hepatitis C virus (HCV)-infected human liver. AIMS: To identity HKGs stable across a wide spectrum of disease in human HCV-infected liver. METHODS: ß-Actin, hypoxanthine phosphoribosyltransferase 1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), splicing factor arginine/serine-rich 4, ß-glucuronidase and 18S ribosomal RNA (18S rRNA) were measured by real-time polymerase chain reaction in liver biopsy tissue. Samples were categorised for inflammation, fibrosis and steatosis, and allocated into groups with mild or severe liver disease. Values were analysed using Spearman's rank correlation, NormFinder, BestKeeper and geNorm programs. RESULTS: All genes performed well in the samples of patients with low disease activity, but HPRT1, ß-actin, GAPDH and 18S rRNA ranked poorly in samples with severe fibrosis or inflammation. CONCLUSIONS: Our results indicate that liver disease affects the expression of common HKGs and that ß-glucuronidase and splicing factor arginine/serine-rich 4 are the most stable HKGs from this group for studies of gene expression in HCV-infected human liver.

Coordinate regulation of metabolic enzymes and transporters by nuclear transcription factors in human liver disease.

BACKGROUND: It has been hypothesised, mainly from studies with animal models of liver disease, that the transport of substrates for metabolic enzymes and their subsequent metabolism and elimination in hepatic bile or blood is co-ordinated, but there is little information on this process in diseased human liver. METHODS: In this study we have measured by reverse transcription polymerase chain reaction (RT-PCR) major genes involved in drug metabolism from UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A9, and UGT2B4) and cytochrome P450 (CYP) families (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), transport (OATP-C, MRP2, MRP3, and MDR1) and major transcription factors (PXR, CAR, HNF1alpha, HNF4alpha, RXR, and AHR) involved in their regulation. Liver biopsy tissue from patients with viral hepatitis was scored for inflammation and fibrosis by the METAVIR system, and separated into groups with mild (A0-1; F0-1, n = 20) or severe (A2-3; F3-4, n = 19) liver disease. Correlation analysis (Spearman rank-test, P < 0.05) was used to identify metabolic enzymes and transporters which shared significant correlation with transcription factors. RESULTS: Our results show an extensive correlation between transcription factors, transporters, and metabolic enzymes. An unexpected finding was that this was substantially greater in the severely diseased liver. Cross-talk between transcription factors was markedly increased in tissue from patients with severe liver disease, particularly between CAR, HNF4alpha, and PXR. CONCLUSION: Our results support the hypothesis of co-ordinate regulation of metabolic enzymes and transporters in diseased human liver, as part of a widespread co-ordinated process under the control of nuclear receptor transcription factors.

Effect of the acute phase response induced by endotoxin administration on the expression and activity of UGT isoforms in rats.

To evaluate the effect of endotoxin administration on the glucuronidation of multiple substrates in a rat model. In addition, the effect of endotoxin treatment on selected UGT mRNA levels in the liver and the kidney was also investigated. Male Sprague-Dawley rats (n= 6) received endotoxin (1.6 mg/kg) by intraperitoneal injection. The animals were sacrificed at 6, 12, 24, 48 hr after treatment, and the liver and the kidneys were harvested. Glucuronidation of various substrates (i.e., acetaminophen, estradiol, testosterone, and morphine) was evaluated using prepared tissue microsomes. Real-Time PCR was used to determine mRNA levels of UGT1A1, 1A6, 2B1, and 2B3 in the harvested organs. UGT1A and UGT2B family isoforms were differentially affected by endotoxin treatment. The greatest reduction in glucuronide formation was observed for estradiol-17-glucuronide (-37%) in hepatic microsomes 48 hr after endotoxin administration. Estradiol-3-glucuronide formation was reduced in liver microsomes (466.3+/-71.5 pmol/min/mg vs. 346.9+/-23.2 pmol/min/mg, p < 0.05) but was not significantly altered in the kidney. Similarly, acetaminophen glucuronide formation was decreased in liver but not kidney microsomes. Significant reductions in glucuronide formation were also observed for morphine (-26%) and testosterone (-30%) in septic rats compared to controls. Endotoxin administration was associated with a time-dependent decrease in UGT1A1, UGT1A6, UGT2B1, and UGT2B3 mRNA levels in liver and kidney tissues. These findings demonstrate that both mRNA and activity of UGT isoforms in rats are decreased following endotoxin treatment, although tissue specific effects do also exist.

UDP glucuronosyltransferase mRNA levels in human liver disease.

The UDP glucuronosyltransferases (UGT) are a family of enzymes in which substrates include drugs, xenobiotics, and products of endogenous catabolism. The main source of most UGT enzymes is the liver, a major organ in the detoxification and inactivation of compounds. Previous studies have indicated that glucuronidation, as measured by pharmacokinetic studies, is relatively spared in liver disease. Because UGT activity toward most substrates is the result of metabolism by different isoforms with overlapping specificities, these studies may not indicate the effect of disease on the levels of individual isoforms. We sought to extend these studies to the measurement of mRNA for individual isoforms in the liver of patients with various forms of liver disease. RNA was extracted from liver tissue samples of patients undergoing clinically necessary percutaneous liver biopsies. UGT mRNA levels for isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17 were determined by real-time reverse transcription-polymerase chain reaction. Biopsies were graded using the Metavir system. Results from patients with low fibrosis or inflammatory scores were compared with those with high scores. We found large interindividual variation in the levels of the various isoforms. This was greatest for UGT2B17. A consistent downward trend, reaching statistical significance for UGT1A4, UGT2B4, and UGT2B7, was observed in samples from patients with high inflammation scores. There was no such correlation with the degree of fibrosis. Our results indicate that hepatic UGT mRNA levels are reduced while the tissue is inflamed, but they are not affected in the noninflamed, chronically diseased liver.