Molecular characterization of Barrett's esophagus at single-cell resolution.

Barrett's esophagus (BE) is categorized, based on morphological appearance, into different stages, which correlate with the risk of developing esophageal adenocarcinoma. More advanced stages are more likely to acquire chromosomal instabilities, but stage-specific markers remain elusive. Here, we performed single-cell DNA-sequencing experiments (scDNAseq) with fresh BE biopsies. Dysplastic BE cells frequently contained chromosomal instability (CIN) regions, and these CIN cells carried mutations corresponding to the COSMIC mutational signature SBS17, which were not present in biopsy-matched chromosomally stable (CS) cells or patient-matched nondiseased control cells. CS cells were predominantly found in nondysplastic BE biopsies. The single-base substitution (SBS) signatures of all CS BE cells analyzed were indistinguishable from those of nondiseased esophageal or gastric cells. Single-cell RNA-sequencing (scRNAseq) experiments with BE biopsies identified two sets of marker genes which facilitate the distinction between columnar BE epithelium and nondysplastic/dysplastic stages. Moreover, histological validation confirmed a correlation between increased CLDN2 expression and the presence of dysplastic BE stages. Our scDNAseq and scRNAseq datasets, which are a useful resource for the community, provide insight into the mutational landscape and gene expression pattern at different stages of BE development.

Fluorescence-Guided Thoracic Duct Dissection in Robotic en Bloc Esophagectomy.

En bloc resection of the thoracic duct compartment enhances adequate lymph node removal and may improve oncologic outcomes in esophagectomy for malignant esophageal diseases. However, it also increases the risk of postoperative chylothorax, with a reported incidence of 5% to 20%. This report describes a technique that facilitates intraoperative identification of the thoracic duct, as well as proximal and distal ligation, during robot-assisted esophagectomy by lymphangiography-guided injection of indocyanine green in the right groin in a patient in the left lateral position. This approach can be swiftly applied at any time during any thoracoscopic procedure using the lateral position when visualization of the thoracic duct anatomy is needed.

Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma.

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P=0.02) and fivefold (P=0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors.

Patient-derived organoids as individual patient models for chemoradiation response prediction in gastrointestinal malignancies.

Chemoradiotherapy (CRT) is an important treatment modality for specific gastrointestinal (GI) cancers, as it has been shown to improve clinical outcomes. Recent developments in the neoadjuvant setting such as wait-and-see strategies for rectal as well as for esophageal cancers have even proven that CRT might be an effective organ-sparing treatment. However, due to molecular heterogeneity, only a subset of patients will show a complete response to CRT, which addresses the need for an individualized treatment approach. In recent years, the demand for more physiologically relevant predictive in vitro models has fostered the development of patient-derived tumor organoids. In this review, we describe the current treatment options for patients with GI cancers who are treated with (neo)adjuvant CRT. Furthermore, we provide an in-depth discussion of the organoid technology in the context of predicting CRT response for GI cancers as well as possible challenges for clinical implementation.

CpG island methylator phenotype predicts progression of malignant melanoma.

PURPOSE: The CpG island methylator phenotype (CIMP) may be associated with development of malignancy through coordinated inactivation of tumor suppressor and tumor-related genes (TRG) and methylation of multiple noncoding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMP pattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigenetic inactivation of TRGs also has been shown in malignant melanoma, we hypothesized the existence of a clinically significant CIMP in cutaneous melanoma progression. EXPERIMENTAL DESIGN: The methylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology (WIF1, TFPI2, RASSF1A, RARbeta2, SOCS1, and GATA4) and a panel of MINT loci (MINT1, MINT2, MINT3, MINT12, MINT17, MINT25, and MINT31) in primary and metastatic tumors of different clinical stages (n=122) was assessed. RESULTS: Here, we show an increase in hypermethylation of the TRGs WIF1, TFPI2, RASSF1A, and SOCS1 with advancing clinical tumor stage. Furthermore, we find a significant positive association between the methylation status of MINT17, MINT31, and TRGs. The methylation status of MINT31 is associated with disease outcome in stage III melanoma. CONCLUSIONS: These findings show the significance of a CIMP pattern that is associated with advancing clinical stage of malignant melanoma. Future prospective large-scale studies may determine if CIMP-positive primary melanomas are at high risk of metastasis or recurrence.

The potential and challenges of patient-derived organoids in guiding the multimodality treatment of upper gastrointestinal malignancies.

The incidence of adenocarcinoma at the gastrooesophageal junction increased over the last years. Curative treatment for patients with upper gastrointestinal (UGI) malignancies, such as oesophageal and gastric tumours, is challenging and requires a multidisciplinary approach. Radical surgical resection with complete lymphadenectomy is the cornerstone of UGI cancer treatment. Combined with peri-operative treatment (i.e. by applying CROSS, EOX or FLOT regimen), the survival is even better than with surgery alone. However, peri-operative treatment is not effective in all patients, and the most effective strategy is a topic of active debate, as is reflected by varying treatment guidelines between countries. UGI cancers are (epi)genetically highly heterogeneous. It is thus not likely that a uniform treatment will benefit all patients equally well. Over recent years, patient-derived organoids (PDOs) gained more and more interest as an in vitro prediction model that may assist as a diagnostic tool in the future to select and eventually optimize the best peri-operative treatments for each patient. PDOs can be derived from endoscopic tumour biopsies, which maintain heterogeneity in culture. They can be rapidly established and expanded in a relatively short time for in vitro drug screening experiments. This review summarizes the clinical and molecular aspects of oesophageal and gastric tumours, as well as the current progress and remaining challenges in the use of PDOs for drug and radiation screens.

Single-Admission, Staged Restoration of Gastrointestinal Continuity After Right Gastroepiploic Artery Damage.

A case of iatrogenic right gastroepiploic artery injury during laparoscopic transhiatal esophagectomy is reported. This case report describes microvascular repair of the right gastroepiploic artery and vein. Subsequent intraoperative decision making with regard to a staged, single-admission successful esophagectomy is discussed. In this case of a single-admission, staged esophagectomy, the gastric conduit was be preserved after transection of the right gastroepiploic artery and vein.

Extended thoracic lymph node dissection in robotic-assisted minimal invasive esophagectomy (RAMIE) for patients with superior mediastinal lymph node metastasis.

BACKGROUND: Robot-assisted surgery may have a role in improving oncological outcomes in esophagectomy. Especially in the anatomical areas in the chest that are more difficult to reach in open surgery (including the superior mediastinum). The dexterity of the robotic instruments aid in performing a more extensive nodal dissection and the precision and detailed vision of the robotic system potentially improves staging, oncological outcomes and reduces complications (i.e., recurrent nerve palsy). In this article, we describe our experience and clinical outcomes in patients treated by robot assisted minimal invasive esophagectomy (RAMIE) in cN+ esophageal cancer patients with positive nodes localized in the superior mediastinum. METHODS: From May 2007-2018, all patients who had involved nodes by either fluor-18-deoxyglucose positron-emission-tomography-computed tomography (FDG-PET-CT) or endoscopic ultrasound (EUS) + fine needle aspiration (FNA) localized in the superior mediastinum (above level Th4/sternal angle) were identified. Patient characteristics, perioperative data, postoperative clinical outcomes/complications and overall survival were prospectively recorded and retrospectively evaluated. RESULTS: Forty patients (48% adenocarcinoma) met our inclusion criteria. All patients underwent a three-stage procedure with cervical anastomosis and 90% of the patients underwent neoadjuvant chemoradiotherapy. Mortality occurred in three patients (7.5%), of which two were caused by severe acute respiratory distress syndrome (ARDS). The most frequent complications were pneumonia (25%), chylothorax (20%), anastomotic leakage (17.5%) and vocal cord paralysis (17.5%) which was grade 1 in 72% of the patients. Radicality rate (R0 resection) was 98% and the average lymph node yield was 24 (range, 9-57). Median overall and disease-free survival was 26 and 17 months, respectively. CONCLUSIONS: RAMIE for esophageal cancer patients with node positive disease in the superior mediastinum is associated with increased mortality/morbidity. Oncological outcome showed excellent lymph node yield, R0 rate and survival was equal compared to patients with lower mediastinal node positive disease.

Robot-assisted minimally invasive esophagectomy (RAMIE) improves perioperative outcomes: a review.

Robotic assisted minimal invasive esophagectomy (RAMIE) is increasingly applied as a clinically and oncologically safe technique in the surgical treatment of esophageal cancer. This review focuses on the advantages and potential opportunities of RAMIE to improve the perioperative and oncological outcomes based on the evidence from current literature. In addition, critical notes on aspects such as procedure duration and costs are addressed in this paper.

Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.

Methylation of p16 and Ras association domain family protein 1a during colorectal malignant transformation.

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60 degrees C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors. On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process.

Improving the outcomes in gastric cancer surgery.

Gastric cancer remains a significant health problem worldwide and surgery is currently the only potentially curative treatment option. Gastric cancer surgery is generally considered to be high risk surgery and five-year survival rates are poor, therefore a continuous strive to improve outcomes for these patients is warranted. Fortunately, in the last decades several potential advances have been introduced that intervene at various stages of the treatment process. This review provides an overview of methods implemented in pre-, intra- and postoperative stage of gastric cancer surgery to improve outcome. Better preoperative risk assessment using comorbidity index (e.g., Charlson comorbidity index), assessment of nutritional status (e.g., short nutritional assessment questionnaire, nutritional risk screening - 2002) and frailty assessment (Groningen frailty indicator, Edmonton frail scale, Hopkins frailty) was introduced. Also preoperative optimization of patients using prehabilitation has future potential. Implementation of fast-track or enhanced recovery after surgery programs is showing promising results, although future studies have to determine what the exact optimal strategy is. Introduction of laparoscopic surgery has shown improvement of results as well as optimization of lymph node dissection. Hyperthermic intraperitoneal chemotherapy has not shown to be beneficial in peritoneal metastatic disease thus far. Advances in postoperative care include optimal timing of oral diet, which has been shown to reduce hospital stay. In general, hospital volume, i.e., centralization, and clinical audits might further improve the outcome in gastric cancer surgery. In conclusion, progress has been made in improving the surgical treatment of gastric cancer. However, gastric cancer treatment is high risk surgery and many areas for future research remain.

The CpG island methylator phenotype: what's in a name?

Although the CpG island methylator phenotype (CIMP) was first identified and has been most extensively studied in colorectal cancer, the term "CIMP" has been repeatedly used over the past decade to describe CpG island promoter methylation in other tumor types, including bladder, breast, endometrial, gastric, glioblastoma (gliomas), hepatocellular, lung, ovarian, pancreatic, renal cell, and prostate cancers, as well as for leukemia, melanoma, duodenal adenocarninomas, adrenocortical carcinomas, and neuroblastomas. CIMP has been reported to be useful for predicting prognosis and response to treatment in a variety of tumor types, but it remains unclear whether or not CIMP is a universal phenomenon across human neoplasia or if there should be cancer-specific definitions of the phenotype. Recently, it was shown that somatic isocitrate dehydrogenase-1 (IDH1) mutations, frequently observed in gliomas, establish CIMP in primary human astrocytes by remodeling the methylome. Interestingly, somatic IDH1 and IDH2 mutations, and loss-of-function mutations in ten-eleven translocation (TET) methylcytosine dioxygenase-2 (TET2) associated with a hypermethylation phenotype, are also found in multiple enchondromas of patients with Ollier disease and Mafucci syndrome, and leukemia, respectively. These data provide the first clues for the elucidation of a molecular basis for CIMP. Although CIMP appears as a phenomenon that occurs in various cancer types, the definition is poorly defined and differs for each tumor. The current perspective discusses the use of the term CIMP in cancer, its significance in clinical practice, and future directions that may aid in identifying the true cause and definition of CIMP in different forms of human neoplasia.

LINE-1 hypomethylation during primary colon cancer progression.

BACKGROUND: Methylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage. METHODOLOGY/PRINCIPAL FINDINGS: DNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05). CONCLUSION/SIGNIFICANCE: By using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.

Identification of a quantitative MINT locus methylation profile predicting local regional recurrence of rectal cancer.

PURPOSE: Risk assessment for locoregional disease recurrence would be highly valuable in preoperative treatment planning for patients undergoing primary rectal tumor resection. Epigenetic aberrations such as DNA methylation have been shown to be significant prognostic biomarkers of disease outcome. In this study, we evaluated the significance of a quantitative epigenetic multimarker panel analysis of primary tumors to predict local recurrence in rectal cancer patients from a retrospective multicenter clinical trial. EXPERIMENTAL DESIGN: Primary tumors were studied from patients enrolled in the trial who underwent total mesorectal excision for rectal cancer (n=325). Methylation levels of seven methylated-in-tumor (MINT) loci were assessed by absolute quantitative assessment of methylated alleles. Unsupervised random forest clustering of quantitative MINT methylation data was used to show subclassification into groups with matching methylation profiles. RESULTS: Variable importance parameters [Gini-Index (GI)] of the clustering algorithm indicated MINT3 and MINT17 (GI, 20.2 and 20.7, respectively) to be informative for patient grouping compared with the other MINT loci (highest GI, 12.2). When using this two-biomarker panel, four different patient clusters were identified. One cluster containing 73% (184 of 251) of the patients was at significantly increased risk of local recurrence (hazard ratio, 10.23; 95% confidence interval, 1.38-75.91) in multivariate analysis, corrected for standard prognostic factors of rectal cancer. This group showed a significantly higher local recurrence probability than patients receiving preoperative radiation (P<0.0001). CONCLUSION: Quantitative epigenetic subclassification of rectal cancers has clinical utility in distinguishing tumors with increased risk for local recurrence and may help tailor treatment regimens for locoregional control.

Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.

PURPOSE: There are no accurate prognostic biomarkers specific for rectal cancer. Epigenetic aberrations, in the form of DNA methylation, accumulate early during rectal tumor formation. In a preliminary study, we investigated absolute quantitative methylation changes associated with tumor progression of rectal tissue at multiple genomic methylated-in-tumor (MINT) loci sequences. We then explored in a different clinical patient group whether these epigenetic changes could be correlated with clinical outcome. PATIENTS AND METHODS: Absolute quantitative assessment of methylated alleles was used to assay methylation changes at MINT 1, 2, 3, 12, 17, 25, and 31 in sets of normal, adenomatous, and malignant tissues from 46 patients with rectal cancer. Methylation levels of these biomarkers were then assessed in operative specimens of 251 patients who underwent total mesorectal excision (TME) without neoadjuvant radiotherapy in a multicenter clinical trial. RESULTS: Methylation at MINT 2, 3, and 31 increased 11-fold (P = .005), 15-fold (P < .001), and two-fold (P = .02), respectively, during adenomatous transformation in normal rectal epithelium. Unsupervised grouping analyses of quantitative MINT methylation data of TME trial patients demonstrated two prognostic subclasses. In multivariate analysis of node-negative patients, this subclassification was the only predictor for distant recurrence (hazard ratio [HR], 4.17; 95% CI, 1.72 to 10.10; P = .002), cancer-specific survival (HR, 3.74; 95% CI, 1.4 to 9.43; P = .003), and overall survival (HR, 2.68; 95% CI, 1.41 to 5.11; P = .005). CONCLUSION: Methylation levels of specific MINT loci can be used as prognostic variables in patients with American Joint Committee on Cancer stage I and II rectal cancer. Quantitative epigenetic classification of rectal cancer merits evaluation as a stratification factor for adjuvant treatment in early disease.

Epigenetic silencing of cyclooxygenase-2 affects clinical outcome in gastric cancer.

PURPOSE: Overexpression of cyclooxygenase-2 (COX-2) in gastric cancer has been shown to enhance tumor progression. We investigated whether silencing by promoter region hypermethylation of the COX-2 gene contributes to disease outcome in gastric cancer. MATERIALS AND METHODS: COX-2 methylation status was initially assessed by capillary array electrophoresis methylation-specific polymerase chain reaction (CAE-MSP) and COX-2 protein expression by immunohistochemistry (IHC) in 40 primary gastric cancer tissues in a pilot study. Prognostic end points of correlative studies of COX-2 methylation status were time to recurrence, overall survival, and standard clinicopathologic features. CAE-MSP analysis was then validated in a second independent gastric cancer population (n = 137). RESULTS: COX-2 methylation was detected in 23% and 28% of the pilot and validation patient groups, respectively. COX-2 expression (IHC) in gastric tumors inversely correlated with COX-2 gene methylation status in the pilot study (P = .02). COX-2 methylation in tumors was significantly associated with lower T, N, and TNM stage in the validation patient group (P = .02, P = .006, and P = .008, respectively). Patients with COX-2 methylated tumors had significantly longer time to recurrence and improved overall survival in a multivariate analysis in the validation patient group (hazard ratio[HR], 0.49; 95% CI, 0.24% to 0.99%; HR, 0.62; 95% CI, 0.38% to 0.99%, respectively). CONCLUSION: Hypermethylation of COX-2 gene promoter was identified as an independent prognostic factor in gastric cancer patients. The results suggest promoter hypermethylation to be an important regulatory mechanism of COX-2 expression in gastric cancer and an important prognostic biomarker.

Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase.

Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with phi29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 x 10(-7), below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested phi29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested phi29 WGA is practically very useful for methylation analysis.