Stephanie C. Hicks

Prediction of missense mutation functionality depends on both the algorithm and sequence alignment employed

Multiple algorithms are used to predict the impact of missense mutations on protein structure and function using algorithm‐generated sequence alignments or manually curated alignments. We compared the accuracy with native alignment of SIFT, Align‐GVGD, PolyPhen‐2, and Xvar when generating functionality predictions of well‐characterized missense mutations (n = 267) within the BRCA1, MSH2, MLH1, and TP53 genes. We also evaluated the impact of the alignment employed on predictions from these algorithms (except Xvar) when supplied the same four alignments including alignments automatically generated by (1) SIFT, (2) Polyphen‐2, (3) Uniprot, and (4) a manually curated alignment tuned for Align‐GVGD. Alignments differ in sequence composition and evolutionary depth. Data‐based receiver operating characteristic curves employing the native alignment for each algorithm result in area under the curve of 78–79% for all four algorithms. Predictions from the PolyPhen‐2 algorithm were least dependent on the alignment employed. In contrast, Align‐GVGD predicts all variants neutral when provided alignments with a large number of sequences. Of note, algorithms make different predictions of variants even when provided the same alignment and do not necessarily perform best using their own alignment. Thus, researchers should consider optimizing both the algorithm and sequence alignment employed in missense prediction. Hum Mutat 32:1–8, 2011.

Development and Validation of a Risk-Stratification Score for Surgical Site Occurrence and Surgical Site Infection after Open Ventral Hernia Repair

BACKGROUND Current risk-assessment tools for surgical site occurrence (SSO) and surgical site infection (SSI) are based on expert opinion or are not specific to open ventral hernia repairs. We aimed to develop a risk-assessment tool for SSO and SSI and compare its performance against existing risk-assessment tools in patients with open ventral hernia repair. STUDY DESIGN A retrospective study of patients undergoing open ventral hernia repair (n = 888) was conducted at a single institution from 2000 through 2010. Rates of SSO and SSI were determined by chart review. Stepwise regression models were built to identify predictors of SSO and SSI and internally validated using bootstrapping. Odds ratios were converted to a point system and summed to create the Ventral Hernia Risk Score (VHRS) for SSO and SSI, respectively. Area under the receiver operating characteristic curve was used to compare the accuracy of the VHRS models against the National Nosocomial Infection Surveillance Risk Index, Ventral Hernia Working Group (VHWG) grade, and VHWG score. RESULTS The rates of SSO and SSI were 33% and 22%, respectively. Factors associated with SSO included mesh implant, concomitant hernia repair, dissection of skin flaps, and wound class 4. Predictors of SSI included concomitant repair, dissection of skin flaps, American Society of Anesthesiologists class ≥ 3, wound class 4, and body mass index ≥ 40. The accuracy of the VHRS in predicting SSO and SSI exceeded National Nosocomial Infection Surveillance and VHWG grade, but was not better than VHWG score. CONCLUSIONS The VHRS identified patients at increased risk for SSO/SSI more accurately than the National Nosocomial Infection Surveillance scores and VHWG grade, and can be used to guide clinical decisions and patient counseling.

On the widespread and critical impact of systematic bias and batch effects in single-cell RNA-Seq data

Single-cell RNA-Sequencing (scRNA-Seq) has become the most widely used high-throughput method for transcription profiling of individual cells. Systematic errors, including batch effects, have been widely reported as a major challenge in high-throughput technologies. Surprisingly, these issues have received minimal attention in published studies based on scRNA-Seq technology. We examined data from five published studies and found that systematic errors can explain a substantial percentage of observed cell-to-cell expression variability. Specifically, we found that the proportion of genes reported as expressed explains a substantial part of observed variability and that this quantity varies systematically across experimental batches. Furthermore, we found that the implemented experimental designs confounded outcomes of interest with batch effects, a design that can bring into question some of the conclusions of these studies. Finally, we propose a simple experimental design that can ameliorate the effect of theses systematic errors have on downstream results.Until recently, high-throughput gene expression technology, such as RNA-Sequencing (RNA-seq) required hundreds of thousands of cells to produce reliable measurements. Recent technical advances permit genome-wide gene expression measurement at the single-cell level. Single-cell RNA-Seq (scRNA-seq) is the most widely used and numerous publications are based on data produced with this technology. However, RNA-Seq and scRNA-seq data are markedly different. In particular, unlike RNA-Seq, the majority of reported expression levels in scRNA-seq are zeros, which could be either biologically-driven, genes not expressing RNA at the time of measurement, or technically-driven, gene expressing RNA, but not at a sufficient level to detected by sequencing technology. Another difference is that the proportion of genes reporting the expression level to be zero varies substantially across single cells compared to RNA-seq samples. However, it remains unclear to what extent this cell-to-cell variation is being driven by technical rather than biological variation. Furthermore, while systematic errors, including batch effects, have been widely reported as a major challenge in high-throughput technologies, these issues have received minimal attention in published studies based on scRNA-seq technology. Here, we use an assessment experiment to examine data from published studies and demonstrate that systematic errors can explain a substantial percentage of observed cell-to-cell expression variability. Specifically, we present evidence that some of these reported zeros are driven by technical variation by demonstrating that scRNA-seq produces more zeros than expected and that this bias is greater for lower expressed genes. In addition, this missing data problem is exacerbated by the fact that this technical variation varies cell-to-cell. Then, we show how this technical cell-to-cell variability can be confused with novel biological results. Finally, we demonstrate and discuss how batch-effects and confounded experiments can intensify the problem.

Evolutionary action score of TP53 identifies high-risk mutations associated with decreased survival and increased distant metastases in head and neck cancer

TP53 is the most frequently altered gene in head and neck squamous cell carcinoma, with mutations occurring in over two-thirds of cases, but the prognostic significance of these mutations remains elusive. In the current study, we evaluated a novel computational approach termed evolutionary action (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk, and validated this system in both in vivo and in vitro models. Patients with high-risk TP53 mutations had the poorest survival outcomes and the shortest time to the development of distant metastases. Tumor cells expressing high-risk TP53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastases. We also documented an association between the presence of high-risk mutations and decreased expression of TP53 target genes, highlighting key cellular pathways that are likely to be dysregulated by this subset of p53 mutations that confer particularly aggressive tumor behavior. Overall, our work validated EAp53 as a novel computational tool that may be useful in clinical prognosis of tumors harboring p53 mutations.

Outcomes of laparoscopic vs open repair of primary ventral hernias

IMPORTANCE More primary ventral hernias (PVHs) are being repaired using the technique of laparoscopic ventral hernia repair (LVHR). Few studies exist comparing the outcomes of LVHR with the outcomes of open ventral hernia repair (OVHR) for PVHs. We hypothesize that LVHR of PVHs is associated with fewer surgical site infections (SSIs) but more hernia recurrences and more clinical cases of bulging (bulging not associated with recurrence or seroma). OBJECTIVE To compare the outcomes of patients who underwent LVHR with the outcomes of patients who underwent OVHR. DESIGN Retrospective study of 532 consecutive patients who underwent an elective PVH repair at a single institution from 2000 to 2010. The outcomes of the 2 procedures were compared using 2 statistical methods. Multivariable logistic regression was used to evaluate the association between outcomes and several independent factors, adjusting for treatment propensity, and the outcomes in the 2 groups of patients were compared using paired univariate analysis. SETTING Michael E. DeBakey VA Medical Center in Houston, Texas. PARTICIPANTS Seventy-nine patients who underwent LVHR and 79 patients who underwent OVHR. MAIN OUTCOMES AND MEASURES The primary outcomes of interest were SSI, hernia recurrence, and bulging. The 2 groups of patients were matched by hernia size, American Society of Anesthesiologists class, age, and body mass index. RESULTS There were 91 patients who underwent an LVHR and 167 patients who underwent an OVHR with mesh, with a median follow-up period of 56 months (range, 1-156 months). Seventy-nine patients with an LVHR were matched to 79 patients with an OVHR. No significant differences in demographic data or confounding factors were detected between the 2 groups. Compared with OVHR, LVHR was significantly associated with fewer SSIs (7.6% vs 34.1%; P < .01) but more clinical cases of bulging (21.5% vs 1.3%; P < .01) and port-site hernia (2.5% vs 0.0%). No differences in recurrence at the site of the hernia repair were observed (11.4% vs 11.4%; P = .99). Propensity score-matched multivariate analysis corroborated that LVHR is associated with more clinical cases of bulging but fewer SSIs. CONCLUSIONS AND RELEVANCE Compared with OVHR of PVHs, LVHR of PVHs is associated with fewer SSIs but more clinical cases of bulging and with the risk of developing a port-site hernia. Further study is needed to clarify the role of LVHR of PVHs and to mitigate the risk of port-site hernia and bulging.

Evolutionary action score of TP53 coding variants is predictive of platinum response in head and neck cancer patients

TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC), with mutations occurring in over two thirds of cases; however, the predictive response of these mutations to cisplatin-based therapy remains elusive. In the current study, we evaluate the ability of the Evolutionary Action score of TP53-coding variants (EAp53) to predict the impact of TP53 mutations on response to chemotherapy. The EAp53 approach clearly identifies a subset of high-risk TP53 mutations associated with decreased sensitivity to cisplatin both in vitro and in vivo in preclinical models of HNSCC. Furthermore, EAp53 can predict response to treatment and, more importantly, a survival benefit for a subset of head and neck cancer patients treated with platinum-based therapy. Prospective evaluation of this novel scoring system should enable more precise treatment selection for patients with HNSCC.

Outcomes and Predictors of Incisional Surgical Site Infection in Stoma Reversal

IMPORTANCE Surgical site infection following stoma reversal (SR) poses a substantial burden to the patient and health care system. Its overall incidence is likely underreported and poorly characterized. Improving our understanding of surgical site infection following stoma reversal may help us identify methods to decrease this complication. OBJECTIVE To evaluate the incidence of surgical site infection (SSI) and identify predictors of SSI following SR. DESIGN A review of computerized hospital records on SR performed from January 1, 2005, until February 27, 2011. SETTING An integrated medical system at the Michael E. DeBakey Veterans Affairs Medical Center. PARTICIPANTS AND INTERVENTION All adults undergoing SR during the study period. MAIN OUTCOME MEASURES Rates of SSI and characteristics of patients with and without SSI were compared. A logistic regression model was developed to identify predictors of SSI. RESULTS One hundred twenty-eight patients underwent SR; 46 patients (36.0%) had an SSI. In comparison with no SSI, the infection was associated with seromas (17.4% vs 2.4%, P = .004), fascial dehiscence (15.2% vs 2.4%, P = .01), intensive care unit admission (34.8% vs 17.1%, P = .03), increased hospital length of stay (20 vs 9 days, P = .02), readmission (32.6% vs 13.4%, P = .01), delayed wound healing (91 vs 66 days, P = .02), and reoperation (32.6% vs 13.4%, P = .01). On multivariate analysis, history of fascial dehiscence (odds ratio, 16.9; 95% CI, 1.94-387), colostomy (5.07; 2.12-13.0), thicker subcutaneous fat (2.02; 1.33-3.21), and black race (0.35; 0.13-0.86) were associated with incisional SSI. There was no significant difference in patient satisfaction or functional status in late follow-up (1-73 months). CONCLUSIONS AND RELEVANCE Surgical site infection is common following SR and is associated with significant morbidity. Four factors are strongly associated with increased risk of SSI in SR: history of fascial dehiscence, thicker subcutaneous fat, colostomy, and white race. Patients with none of these risk factors had a 0% SSI risk; patients with all 4 risk factors had a 100% risk of SSI.

Abdominal reoperation and mesh explantation following open ventral hernia repair with mesh

BACKGROUND This study sought to identify the incidence, indications, and predictors of abdominal reoperation and mesh explantation following open ventral hernia repair with mesh (OVHR). METHODS A retrospective cohort study of all patients at a single institution who underwent an OVHR from 2000 to 2010 was performed. Patients who required subsequent abdominal reoperation or mesh explantation were compared with those who did not. Reasons for reoperation were recorded. The 2 groups were compared using univariate and multivariate analysis (MVA). RESULTS A total of 407 patients were followed for a median (range) of 57 (1 to 143) months. Subsequent abdominal reoperation was required in 69 (17%) patients. The most common reasons for reoperation were recurrence and surgical site infection. Only the number of prior abdominal surgeries was associated with abdominal reoperation on MVA. Twenty-eight patients (6.9%) underwent subsequent mesh explantation. Only the Ventral Hernia Working Group grade was associated with mesh explantation on MVA. CONCLUSIONS Abdominal reoperation and mesh explantation following OVHR are common. Overwhelmingly, surgical complications are themost common causes for reoperation and mesh explantation.

quantro: a data-driven approach to guide the choice of an appropriate normalization method

Normalization is an essential step in the analysis of high-throughput data. Multi-sample global normalization methods, such as quantile normalization, have been successfully used to remove technical variation. However, these methods rely on the assumption that observed global changes across samples are due to unwanted technical variability. Applying global normalization methods has the potential to remove biologically driven variation. Currently, it is up to the subject matter experts to determine if the stated assumptions are appropriate. Here, we propose a data-driven alternative. We demonstrate the utility of our method (quantro) through examples and simulations. A software implementation is available from http://www.bioconductor.org/packages/release/bioc/html/quantro.html.

Outcomes with Porcine Acellular Dermal Matrix versus Synthetic Mesh and Suture in Complicated Open Ventral Hernia Repair

BACKGROUND Mesh reinforcement as part of open ventral hernia repair (OVHR) has become the standard of care. However, there is no consensus on the ideal type of mesh to use. In many clinical situations, surgeons are reluctant to use synthetic mesh. Options in these complicated OVHRs include suture repair or the use of biologic mesh such as porcine acellular dermal matrix (PADM). There has been a paucity of controlled studies reporting long-term outcomes with biologic meshes. We hypothesized that compared with synthetic mesh in OVHR, PADM is associated with fewer surgical site infections (SSI) but more seromas and recurrences. Additionally, compared with suture repair, we hypothesized that PADM is associated with fewer recurrences but more SSIs and seromas. METHODS A retrospective study was performed of all complicated OVHRs performed at a single institution from 2000-2011. All data were captured from the electronic medical records of the service network. Data were compared in two ways. First, patients who had OVHR with PADM were case-matched with patients having synthetic mesh repairs on the basis of incision class, Ventral Hernia Working Group (VHWG) grade, hernia size, American Society of Anesthesiologists (ASA) class, and emergency status. The PADM cases were also matched with suture repairs on the basis of incision class, hernia grade, duration of the operation, ASA class, and emergency status. Second, we developed a propensity score-adjusted multi-variable logistic regression model utilizing internal resampling to identify predictors of primary outcomes of the overall cohort. The U.S. Centers for Disease Control and Prevention (CDC) definition of SSI was utilized; seromas and recurrences were defined and tracked similarly for all patients. Data were analyzed using the McNemar, X(2), paired two-tailed Student t, or Mann-Whitney U test as appropriate. RESULTS A total of 449 complicated OVHR cases were reviewed for a median follow up of 61 mos (range 1-143 mos): 94 patients had PADM repairs, whereas 154 patients underwent synthetic mesh repairs, and 201 had suture repairs. The 40 PADM repairs were matched to synthetic repairs and 59 were matched to suture repairs. The PADM repairs that could not be well matched (n=54 unmatched for synthetic repairs, 35 unmatched for suture repairs) were characterized generally by larger hernias, VHWG grades of 3 or 4, and incision class 3 or 4 with longer operative durations and more ASA class 4 cases. The patients were well matched. Comparing PADM with synthetic mesh, there was no difference in SSI (20% vs. 35%; p=0.29), seromas (32.5% vs. 15%; p=0.17), mesh explantations (5% vs. 15%, p=0.28), readmissions within 90 d (37.5% vs. 45%; p=1.00), or recurrence (8.5% vs. 22.5%; p=0.15). Compared with suture repair, patients with PADM had fewer recurrences (11.9% vs. 33.9%; p<0.01) and more seromas (32.2% vs. 10.2%; p=0.02), but a similar number of SSIs (23.7% vs. 39.0%; p=0.19) and 90-d readmissions (35.6% vs. 39.0%; p=0.88). Propensity score-adjusted multi-variable logistic regression of the entire cohort corroborated the results of the case-matched patients. CONCLUSIONS The PADM repair of complicated OVHR resulted in fewer recurrences, more seromas, and no difference in SSI compared with suture repair. Although no reduction in SSI was identified with the use of PADM rather than synthetic mesh or suture for OVHR, the meaning of this finding is unclear, as this case-controlled study was underpowered and limited by selection bias. According to our data, 280 patients would have been needed to identify a clinically significant difference in the primary outcome of SSI as well as secondary outcomes of mesh explantation and recurrence (α=0.05; β=0.20). A randomized trial is warranted to compare PADM with synthetic mesh in complicated OVHR.