Dustin B. Manders

The impact of reference gene selection in quantification of gene expression levels in guinea pig cervical tissues and cells

BackgroundAccurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix.ResultsGene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes.ConclusionsWe recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells from guinea pig.

Primary iliopsoas abscess and pregnancy.

BACKGROUND: Iliopsoas abscesses, a rare complication in pregnancy, typically present as a secondary infection due to direct spread of another nearby infection. CASE: A 30-year-old multigravid patient with a 10-year history of intravenous heroin use and chronic lower-back pain, presented at 23 0/7 weeks of gestation with multiple cutaneous abscesses. As her back pain worsened during antepartum hospitalization, a primary iliopsoas abscess with underlying osteomyelitis was identified requiring percutaneous computed tomography–guided drainage along with extended-course intravenous antibiotics. After treatment and methadone detoxification, her pregnancy resulted in a successful vaginal delivery. CONCLUSION: Primary iliopsoas abscess should be considered in the differential diagnosis of low-back pain among high-risk pregnant women. Clinicians should be vigilant in identifying high-risk factors and aggressive in treating this rare and potentially fatal complication.

Factors associated with clinical trial screening failures in gynecologic oncology

OBJECTIVE Low enrollment of adult cancer patients in clinical trials is an ongoing challenge in cancer research. We sought to determine factors associated with clinical trial screening failures in women with gynecologic malignancies at a large urban university health system. METHODS A retrospective review was conducted of women with gynecologic malignancies who presented to an urban university system between 12/2009 and 12/2012. Data collected included demographic, clinico-pathologic and trial-related factors, as well as reasons for non-participation. RESULTS Two hundred twenty-one patients were eligible for a clinical trial. Of these, 44% participated while 56% did not. There were more screening failures when trials were offered at the time of primary treatment than at recurrence (62% vs. 38%, p=0.001). There was no significant difference in participation based on age, ethnicity, hospital setting, payor status, family history, comorbidities, prior treatment, substance abuse, recent surgery or trial type. Of the non-participants, 62% declined the study due to perceived harm and 10% due to socio-economic barriers while 20% were excluded due to co-morbidities and 8% due to noncompliance. CONCLUSIONS Significantly more screening failures for clinical trials occurred when trials were offered at the time of primary treatment. The majority of patients declined based on perceived harm from enrolling in a clinical trial, although 20% of eligible patients were not offered enrollment despite not meeting any exclusion criteria. Our findings underscore the importance of appropriate counseling when offering clinical trials, as well as overcoming physician biases in deciding who is an appropriate candidate.

Dysregulation of fibulin-5 and matrix metalloproteases in epithelial ovarian cancer

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

Response to “Towards implementation of sexual healthcare”

Response to comment from Albers et al.: We thank Albers et al. for their interest and review of our recent publication. We agree with their consensus that despite sexual dysfunction being a prevalent problem amongst cervical cancer patients, health care providers rarely broach these issues. Additionally, these authors provide findings that even if clinicians report receiving adequate sexual healthcare training, they are still hesitant to initiate relevant discussions with patients (Jonsdottir et al., 2016). Accordingly, Albers et al. recognize that a lack of knowledge may not be the only contributing factor to this reluctance. As demonstrated by our findings, various personal motivations and factors of providers may contribute as well. In contrast to our findings that clinicians with more years of experience are more likely to agree that discussing sexual issues with patients may interfere with the patient-provider relationship (Bedell et al., 2017), Albers et al. conclude that more experienced surgical oncologists discuss these topics more often. One offered explanation for this discrepancy by these authors is the notion that a modeled, multi-disciplinary approach, that merges sexual healthcare into clinical practice, is not available for example. Suitably, these authors advocate for the development of methods that may enhance sexual care in the current clinical setting. Our authors agree with this sentiment, as an enhanced focus on the practical implementations of sexual healthcare into clinical practice will ultimately serve to support an integrated focus on this important issue. It is encouraging and exciting to see other providers who view this as a priority. Thank your for your comment and we look forward to viewing your future research.

Third-line Salvage Chemotherapy for Recurrent Carcinoma of the Cervix is Associated With Minimal Response Rate and High Toxicity

Background: Metastatic and recurrent cervical cancer is rarely a curable disease. Systemic chemotherapy is typically recommended for treatment based on clinical trials in the first-line or second-line setting. Rare patients who progress through 2 salvage regimens will have the performance status, medical ability, and desire to continue cytotoxic therapy. For these patients, there are no data to provide effective counseling regarding expected response rates (RRs) and toxicities. We sought to review our experience with this patient population. Methods: A single institution review was performed of all patients treated for cervical cancer between January 1, 2000 and June 30, 2013. Eligible patients were those who received at least 3 unique salvage chemotherapy regimens following primary surgery or radiation. RRs, survival statistics and toxicities were evaluated. Results: Twenty-three of 710 (3.2%) patients treated for cervical cancer met eligibility criteria. Nineteen received 2 or more cycles of a third-line regimen and were assessed for response and progression-free survival. The remainder were included in analysis of overall survival and toxicity. The RR to third-line chemotherapy was 10% (1 complete, 1 partial). An additional 27% achieved stable disease. In total, 57% suffered a grade 3 or 4 toxicity. The progression-free survival from the beginning of third-line therapy was 3.8 months, and the overall survival was 7.4 months. Conclusions: Patients eligible to receive third-line chemotherapy for metastatic and recurrent cervical cancer can expect minimal benefit at the cost of significant toxicity. Quality of life considerations should be of paramount importance when counseling regarding the risks and benefits of further cytotoxic therapy.

Locally Advanced Cervical Cancer: Outcomes With Variable Adherence to Treatment.

Objective: Adherence to treatment regimen and schedule is recommended to improve control of disease and overall survival (OS) in locally advanced cervical cancer. However, treatment-related toxicities and patient and physician factors all impact timely completion of treatment. We sought to correlate adherence to treatment plan with survival and toxicities of patients treated for locally advanced cervical cancer. Materials and Methods: A retrospective review of patients treated for advanced cervical cancer at our institution between 2003 and 2011 was performed. Demographics, clinicopathologic variables, treatment, and disease outcomes were collected. Endpoints of disease outcome were disease-free survival and OS. Statistical analyses were performed using the Kaplan-Meier method, log-rank test, and Cox regression analysis. Results: A total of 162 patients met the inclusion criteria and were included in study analysis. A total of 95% of patients were treated with both radiation and concurrent chemotherapy. Mean radiation dose to point A was 72 Gy. In total, 77% had complete response to primary therapy. Severe (grade 3/4) late radiation toxicities were seen in 10.5% of patients. Stage and total radiation dose to point A were significant predictors of survival for the entire cohort. Among patients receiving at least 72 Gy and brachytherapy, duration of treatment was significantly associated with both disease-free survival and OS. Conclusions: Adherence to both optimal treatment time and radiation dose is significantly associated with improved survival. Total radiation dose is an independent predictor of survival among patients with locally advanced cervical cancer.