Correction: Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

[This corrects the article DOI: 10.1371/journal.pone.0179501.].

Post-cholecystectomy Gallstone Ileus.

Gallstone ileus is an unusual complication of cholelithiasis. Classically, a stone is impacted at the terminal ileum originating from a cholecystoduodenal fistula. Exceptions to this pathophysiology have been noted at each step. In this systematic review, we document a comprehensive review of postcholecystectomy gallstone ileus inclusive of 49 separate cases and report 8 different mechanisms leading to this unusual complication. The most common mechanism is a lost stone during cholecystectomy that then erodes through the intestinal wall leading to bowel obstruction. Our review showed an older, female predominance (64.0%) at an average age of 68.0 years, patients typically had a burden of comorbidities. Delay in diagnosis was common (64% of cases) with the correct diagnosis made in 37.5% of patients during admission. Pneumobilia was commonly reported (29.0%). There was a wide range in the amount of time between cholecystectomy and gallstone ileus, from 10 days to 50 years (mean 12.4 years). Postcholecystectomy gallstone ileus is an unusual complication of cholelithiasis, which mandates surgery. Retrieval of stones should be undertaken if they are spilled during cholecystectomy. Owing to the increasing age of the American patient population, it is likely that a higher number of patients with this condition will be encountered.

Simulation-based mastery learning significantly reduces gender differences on the Fundamentals of Endoscopic Surgery performance exam.

BACKGROUND: Analysis of the Fundamentals of Endoscopic Surgery (FES) performance exam showed higher scores for men than women. Gender differences have been reduced with task-specific practice. We assessed the effect of simulation-based mastery learning (SBML) on FES performance exam differences by gender. METHODS: Forty-seven surgical trainees [29 men (m), 18 women (w)] completed a SBML curriculum and were assessed by FES. Fourteen trained on the GI Mentor 2, 18 on the Endoscopy Training System, and 15 using the Surgical Training for Endoscopic Proficiency curriculum. Performance of male and female trainees was compared. RESULTS: On the pre-training assessment, there were large differences between genders in FES pass rates (m 77%, w 15%, p < 0.001), total scores (m 69 ± 11, w 50 ± 12; p < 0.001), and in four of five FES sub-task scores (Navigation, m 73 ± 19, w 55 ± 22, p = 0.02; Loop reduction, m 34 ± 29, w 14 ± 22, p = 0.02; Retroflexion, m 81 ± 17, w 47 ± 27, p < 0.001; Targeting, m 89 ± 10, w 66 ± 23, p = 0.002). No differences were discernible post training (Pass rate, m 100%, w 94%, p = 0.4; Total score, m 77 ± 8, w 72 ± 12, p = 0.2; Navigation, m 91 ± 13, w 80 ± 13, p = 0.009; Loop reduction, m 49 ± 26, w 46 ± 36, p = 0.7; Retroflexion, m 82 ± 18, w 81 ± 15, p = 0.9; Targeting, m 92 ± 15, w 86 ± 12, p = 0.12). Time needed to complete curricula was not discernably different by gender (m 3.8 ± 1.7 h, w 5.0 ± 2.6 h, p = 0.17). CONCLUSIONS: Gender-based differences are nearly eliminated through task-specific SBML training. This lends further evidence to the validity argument for the FES performance exam as a measure of basic endoscopic skills.

Proficiency-based preparation significantly improves FES certification performance.

BACKGROUND: The Fundamentals of Endoscopic Surgery (FES) certification has recently been mandated by the American Board of Surgery but best methods for preparing for the exam are lacking. Our previous work demonstrated a 40% pass rate for PGY5 residents in our program. The purpose of this study was to determine the effectiveness of a proficiency-based skills and cognitive curriculum for FES certification. METHODS: Residents who agreed to participate (n = 15) underwent an orientation session, followed by skills pre-testing using three previously described models (Trus, Operation targeting task, and Kyoto) as well as the actual FES skills exam (vouchers provided by the FES committee). Participants then trained to proficiency on all three models for the skills curriculum and completed the FES online didactic material for the cognitive curriculum. Finally, participants post-tested on the models and took the actual FES certification exam. Values are mean ± SD; p < 0.05 was considered significant. RESULTS: Of 15 residents who participated, 8 (53%) passed the FES skills exam at baseline. Participants required 2.7 ± 1.3 h to achieve proficiency on the models and approximately 3 h to complete the cognitive curriculum. At post-test, 14 (93%, vs. pre-test 53%, p = 0.041) passed the FES skills exam. 14 (93%) passed the FES cognitive exam and 13/15 (87%) passed both the skills and cognitive exam and achieved FES certification. CONCLUSIONS: Our traditional clinical endoscopy curricula were not sufficient for senior residents to pass the FES exam. Implementation of a proficiency-based flexible endoscopy curriculum using bench-top models and the FES online materials was feasible and effective for the majority of learners. Importantly, with a modest amount of additional training, 87% of our trainees were able to pass the FES examination, which represents a significant improvement for our program. We expect that additional refinements of this curriculum may yield even better results for preparing future residents for the FES examination.

Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

Long-term outcomes in treatment of retroperitoneal sarcomas: A 15 year single-institution evaluation of prognostic features.

BACKGROUND: Retroperitoneal sarcomas are connective tissue tumors arising in the retroperitoneum. Surgical resection is the mainstay of treatment. Debate has arisen over extent of resection, changes in histological classification/grading, and interest in incorporating radiotherapy. Therefore, we reviewed our institution's experience to evaluate prognostic factors. METHODS: Retrospective chart review of all primary RPS patients at Johns Hopkins Hospital from 1994 to 2010. Histologic diagnosis and grading were re-evaluated with current criteria. Prognostic factors for survival, and recurrence were assessed. RESULTS: One hundred thirty-one primary RPS patients met inclusion criteria. Median survival for patients who undergo en-bloc resection to negative margins (R0/R1) is 81.7 months. Surgical margins and grade were the most important factors for survival along with age, gender, presence of metastases and resection of ≥5 organs. Five-year survival for R0/R1 resection was 60%, similar to compartmental resection. Radiotherapy significantly decreased local recurrence (P = 0.026) on multivariate analysis. Grade in leiomyosarcomas and dedifferentiation in liposarcomas dictated patterns of local versus distal recurrence. CONCLUSIONS: En bloc surgical resection to R0/R1 margins remains the cornerstone of therapy and provides comparable outcomes to compartmental resections. Grade remains important for prognosis, and histology dictates recurrence patterns. Radiotherapy appears promising for local control and warrants further investigation. J. Surg. Oncol. 2016;114:56-64. © 2016 Wiley Periodicals, Inc.

IGFBP-3 Gene Methylation in Primary Tumor Predicts Recurrence of Stage II Colorectal Cancers.

OBJECTIVES: To evaluate the influence of IGFBP-3 methylation on recurrence in patients with stage II colorectal cancer (CRC) from 2 independent cohorts. BACKGROUND: The relationship between IGFBP-3 methylation in primary tumors (PTs) or lymph nodes (LNs) and risk of recurrence in patients with stage II CRC treated with surgery alone is unknown. METHODS: IGFBP-3 methylation of DNA from 115 PTs and 1641 LNs in patients with stage II CRC from 2 independent cohorts was analyzed. Forty patients developed recurrence, whereas 75 matched patients remained recurrence free for more than 2 years after surgery. Cox proportional hazard models were used to calculate hazard ratios (HRs) of recurrence, adjusted for patient and tumor characteristics. RESULTS: Methylation of IGFBP-3 in PTs was identified to be significantly associated with risk of recurrence in the training set. The signature was tested in a validation set and classified 40.7% of patients as high risk. Five-year recurrence-free survival rates were 76.4% and 58.3% for low- and high-risk patients, respectively, with an HR of 2.21 (95% confidence interval, 1.04-4.68; P = 0.039). In multivariate analysis, the signature remained the most significant prognostic factor, with an HR of 2.40 (95% confidence interval, 1.10-5.25; P = 0.029). A combined analysis of 1641 LNs from the 2 sets identified IGFBP-3 methylation in LNs was not associated with risk of recurrence. CONCLUSIONS: Detection of IGFBP-3 methylation in PTs, but not in LNs, provides a powerful tool for the identification of patients with stage II CRC at high risk of recurrence.

The promise of methylation on beads for cancer detection and treatment.

Despite numerous technical hurdles, the realization of true personalized medicine is becoming a progressive reality for the future of patient care. With the development of new techniques and tools to measure the genetic signature of tumors, biomarkers are increasingly being used to detect occult tumors, determine the choice of treatment and predict outcomes. Methylation of CpG islands at the promoter region of genes is a particularly exciting biomarker as it is cancer-specific. Older methods to detect methylation were cumbersome, operator-dependent and required large amounts of DNA. However, a newer technique called methylation on beads has resulted in a more uniform, streamlined and efficient assay. Furthermore, methylation on beads permits the extraction and processing of miniscule amounts of methylated tumor DNA in the peripheral blood. Such a technique may aid in the clinical detection and treatment of cancers in the future.

Immune regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common human epithelial cancers.

Epigenetic therapy is emerging as a potential therapy for solid tumors. To investigate its mechanism of action, we performed integrative expression and methylation analysis of 63 cancer cell lines (breast, colorectal, and ovarian) after treatment with the DNA methyltransferase inhibitor 5-azacitidine (AZA). Gene Set Enrichment Analysis demonstrated significant enrichment for immunomodulatory pathways in all three cancers (14.4-31.3%) including interferon signaling, antigen processing and presentation, and cytokines/chemokines. Strong upregulation of cancer testis antigens was also observed. An AZA IMmune gene set (AIMs) derived from the union of these immunomodulatory pathway genes classified primary tumors from all three types, into "high" and "low" AIM gene expression subsets in tumor expression data from both TCGA and GEO. Samples from selected patient biopsies showed upregulation of AIM genes after treatment with epigenetic therapy. These results point to a broad immune stimulatory role for DNA demethylating drugs in multiple cancers.

CpG island methylator phenotype and its association with malignancy in sporadic duodenal adenomas.

CpG island methylator phenotype (CIMP) has been found in multiple precancerous and cancerous lesions, including colorectal adenomas, colorectal cancers, and duodenal adenocarcinomas. There are no reports in the literature of a relationship between CIMP status and clinicopathologic features of sporadic duodenal adenomas. This study sought to elucidate the role of methylation in duodenal adenomas and correlate it with KRAS and BRAF mutations. CIMP+ (with more than 2 markers methylated) was seen in 33.3% of duodenal adenomas; 61% of these CIMP+ adenomas were CIMP-high (with more than 3 markers methylated). Furthermore, CIMP+ status significantly correlated with older age of patients, larger size and villous type of tumor, coexistent dysplasia and periampullary location. MLH1 methylation was seen in 11.1% of duodenal adenomas and was significantly associated with CIMP+ tumors, while p16 methylation was an infrequent event. KRAS mutations were frequent and seen in 26.3% of adenomas; however, no BRAF mutations were detected. Furthermore, CIMP-high status was associated with larger size and villous type of tumor and race (non-white). These results suggest that CIMP+ duodenal adenomas may have a higher risk for developing malignancy and may require more aggressive management and surveillance.

Novel methylation biomarker panel for the early detection of pancreatic cancer.

PURPOSE: Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here, we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer. EXPERIMENTAL DESIGN: We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of eight promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis, including methylation and expression. We used a nanoparticle-enabled methylation on beads (MOB) technology to detect early-stage pancreatic cancers by analyzing DNA methylation in patient serum. RESULTS: We identified two novel genes, BNC1 (92%) and ADAMTS1 (68%), that showed a high frequency of methylation in pancreatic cancers (n = 143), up to 100% in PanIN-3 and 97% in stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n = 42) from patients with pancreatic cancer, with a sensitivity for BNC1 of 79% [95% confidence interval (CI), 66%-91%] and for ADAMTS1 of 48% (95% CI, 33%-63%), whereas specificity was 89% for BNC1 (95% CI, 76%-100%) and 92% for ADAMTS1 (95% CI, 82%-100%). Overall sensitivity using both markers is 81% (95% CI, 69%-93%) and specificity is 85% (95% CI, 71%-99%). CONCLUSIONS: Promoter DNA methylation of BNC1 and ADAMTS1 is a potential biomarker to detect early-stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease.

Extrathoracic location and "borderline" histology are associated with recurrence of solitary fibrous tumors after surgical resection.

BACKGROUND: Most solitary fibrous tumors (SFTs) are cured by complete resection, but many recurrent and metastatic SFTs do not respond to treatment and are fatal. Malignant histology, defined by England's pathologic criteria, is strongly associated with recurrence, but some benign SFTs still behave aggressively. Several studies have suggested that extrathoracic SFTs have a worse prognosis. We reviewed thoracic and extrathoracic SFTs from our institution to determine if extrathoracic location is associated with recurrence, independent of malignant histology. METHODS: With IRB approval, we retrieved patient pathology reports from the Johns Hopkins Surgical Pathology database between 1991 and 2011 and included 83 SFT patients in our analysis. Patient history and outcomes were obtained from the medical record and primary care physicians. Predictors of recurrence were analyzed by univariate and multivariate analysis and survival determined by the Kaplan-Meier method. RESULTS: Of the 83 patients, 59 had extrathoracic SFTs in neurologic (n = 24), extremity or head/neck (n = 13), or visceral/intraabdominal (n = 22) sites. A total of 74 SFTs were classified benign and 9 as malignant. Of the 14 recurrences, 13 occurred in extrathoracic SFTs; only 7 were classified as malignant. Multivariate analysis confirmed that malignant histology had the strongest association with recurrence, but extrathoracic location also independently predicted recurrence. A total of 20 benign SFTs possessed 1 or more of England's criteria but to an insufficient degree for malignant classification. These "borderline" SFTs were more likely to recur than benign SFTs without these features. CONCLUSIONS: Extrathoracic and "borderline" SFTs with any of England's criteria have a higher risk of recurrence.

KRAS G>A mutation favors poor tumor differentiation but may not be associated with prognosis in patients with curatively resected duodenal adenocarcinoma.

KRAS mutations have been found in duodenal adenocarcinomas and may have prognostic significance. The purpose of this study was to classify clinicopathological characteristics, microsatellite instability and KRAS mutations and identify possible prognostic role of KRAS mutations in duodenal adenocarcinomas. Demographics, tumor characteristics and survival were recorded for 78 patients with duodenal adenocarcinomas (Stages I-III). KRAS mutations were detected in 27 (34.6%) cases, of which the majority (74.1%) were G>A transitions. Multivariate logistic regression analysis showed that KRAS G>A mutation was significantly associated with late stage (p = 0.025) and poor tumor differentiation (p = 0.035), when compared with wild-type and other than G>A mutations. KRAS G>A mutation carriers were at increased risk for distant relapse (p = 0.022) and had significantly shorter overall survival (OS; log-rank p = 0.045) and a trend toward shorter relapse-free survival (RFS; log-rank p = 0.062) when compared with those who did not carry the KRAS G>A mutation. In multivariate analyses, there was a significant correlation between ≥ 3 positive lymph nodes and poor OS (p < 0.001) and RFS (p = 0.001) and KRAS G>A mutation carriers demonstrated no effect on clinical outcome. In conclusion, KRAS G>A mutation correlates significantly with late stage and poor tumor differentiation in duodenal adenocarcinoma. Among patients who undergo a curative resection of duodenal adenocarcinoma, KRAS G>A mutation carriers will more likely experience distant relapse but may not exhibit a poor prognosis. The number of positive lymph nodes should be incorporated in future staging systems.

CpG island methylator phenotype-positive tumors in the absence of MLH1 methylation constitute a distinct subset of duodenal adenocarcinomas and are associated with poor prognosis.

PURPOSE: Little information is available on genetic and epigenetic changes in duodenal adenocarcinomas. The purpose was to identify possible subsets of duodenal adenocarcinomas based on microsatellite instability (MSI), DNA methylation, mutations in the KRAS and BRAF genes, clinicopathologic features, and prognosis. EXPERIMENTAL DESIGN: Demographics, tumor characteristics, and survival were available for 99 duodenal adenocarcinoma patients. Testing for KRAS and BRAF mutations, MSI, MLH1 methylation, and CpG island methylator phenotype (CIMP) status was conducted. A Cox proportional hazard model was built to predict survival. RESULTS: CIMP(+) was detected in 27 of 99 (27.3%) duodenal adenocarcinomas and was associated with MSI (P = 0.011) and MLH1 methylation (P < 0.001), but not with KRAS mutations (P = 0.114), as compared with CIMP(-) tumors. No BRAF V600E mutation was detected. Among the CIMP(+) tumors, 15 (55.6%) were CIMP(+)/MLH1-unmethylated (MLH1-U). Kaplan-Meier analysis showed that tumors classified by CIMP, CIMP/MLH1 methylation status, or CIMP/MSI status could predict overall survival (OS; P = 0.047, 0.002, and 0.002, respectively), whereas CIMP/MLH1 methylation status could also predict time-to-recurrence (TTR; P = 0.016). In multivariate analysis, CIMP/MLH1 methylation status showed a significant prognostic value in both OS (P < 0.001) and TTR (P = 0.023). Patients with CIMP(+)/MLH1-U tumors had the worst OS and TTR. CONCLUSIONS: Our results showed existence of CIMP in duodenal adenocarcinomas. The combination of CIMP(+)/MLH1-U seems to be independently associated with poor prognosis in patients with duodenal adenocarcinomas. This study also suggests that BRAF mutations are not involved in duodenal tumorigenesis, MSI, or CIMP development.

Epithelioid sarcoma: one institution's experience with a rare sarcoma.

BACKGROUND: Epithelioid sarcomas (ES) are extremely rare soft tissue sarcomas. As such, their clinical behavior and response to treatment are poorly described in the literature. METHODS: We queried the centralized cancer registry and pathology archives at the Johns Hopkins Medical Institution and identified 22 patients with a diagnosis of ES. We excluded two patients because of inadequate data. A pathologist reviewed patient charts and reexamined available histological slides. This study was performed with institutional review board approval. RESULTS: The median age at diagnosis was 27.8 y; most patients (75%) were male. Regional lymph node metastases were present in 10% of patients at presentation. The majority of tumors (57.9%) recurred and 35% recurred more than once, although the number of recurrences did not affect survival (P = 0.48). Patients did not experience a decrease in time to recurrence with increasing number of resections. The median time between resection and recurrence was 1.23 y and the maximum was 18.8 y. Median overall survival was 56.2 mo and 5-y survival was 92%. CONCLUSIONS: Our study reveals that ES is an extremely rare tumor with a protracted and recurrent course, but overall survival may be more favorable than in the past. Patients benefit from aggressive and repeated resection. Epithelioid sarcoma is unique because it metastasizes to regional nodal basins. Extended surveillance is indicated, because recurrences can appear after decades of quiescence.

DNA methylation biomarker candidates for early detection of colon cancer.

Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (>80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine™ website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.