Multiregion sequencing and subclonal analysis reveal intratumoral heterogeneity in esophageal squamous cell carcinoma.

PURPOSE: The aim of this study was to investigate intratumoral genomic heterogeneity and subclonal structure of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Multiregion whole-exome sequencing was performed on 24 surgically acquired tumor samples from five untreated ESCC patients collected in 2019 to determine the heterogeneity of mutational landscape within tumors. Phylogenetic analysis and mutation process analysis were used to explore the distribution and dynamic changes of mutation spectrum, and subclone analysis was used to explore the subclonal composition and spatial structure of ESCC. RESULTS: An average of 60.2% of mutations were found heterogenous. TP53 and NOTCH1 mutations were confirmed to be early events, and mutations unique in different tumor regions showed a pattern of branching evolution. A large proportion of mutations were associated with abnormal activity of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family, and significant differences in mutation types between trunk and branch variants were found. Subclonal structure exhibited spatial correspondence and spatial limitations, and different genomic features were characterized between close and distant clones. CONCLUSIONS: There is significant intratumoral genomic heterogeneity in the five ESCCs, and their subclonal structure is related to spatial locations.

High expression of PDGFA predicts poor prognosis of esophageal squamous cell carcinoma.

ABSTRACT: Platelet-derived growth factor A (PDGFA), the most known member of PDGF family, plays a crucial role in occurrence and progression of different tumors. However, PDGFA expression and its clinical significance in esophageal squamous cell carcinoma (ESCC) are not clear. The present study aimed to assess the expression and prognostic value of PDGFA in ESCC.The Gene Expression Omnibus databases (GSE53625, GSE23400, and GSE67269) and fresh clinical samples were employed for detecting PDGFA messenger RNA expression in ESCC. The associations of PDGFA expression with clinicopathological characteristics were evaluated by chi-square test. Kaplan-Meier analysis and Cox proportional hazard regression model were performed to determine the prognostic value of PDGFA in ESCC patients. PDGFA-related signaling pathways were defined by gene set enrichment analysis based on Gene Expression Omnibus databases.The PDGFA messenger RNA expression was upregulated in ESCC tissues compared with paired adjacent noncancerous tissues (P < .05) and was positively correlated with T stage (P < .05). Kaplan-Meier survival analysis suggested that ESCC patients with high PDGFA expression were associated with poorer overall survival compared to those with low PDGFA expression (P < .05), especially in advanced T stage (P < .05). Cox analyses showed that high expression of PDGFA was an independent predictor for poor prognosis in ESCC patients. Gene set enrichment analysis identified 3 signaling pathways (extracellular matrix receptor interaction, focal adhesion, and glycosaminoglycan biosynthesis chondroitin sulfate) that were enriched in PDGFA high expression phenotype (all P < .01).PDGFA may serve as an oncogene in ESCC and represent an independent molecular biomarker for prognosis of ESCC patients.

MicroRNA­485­5p suppresses the progression of esophageal squamous cell carcinoma by targeting flotillin­1 and inhibits the epithelial­mesenchymal transition.

As esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed cancers in Asia, it is crucial to uncover its underlying molecular mechanisms that support its development and progression. Several articles have reported that microRNA (miR)­485­5p inhibits the malignant phenotype in a number of cancer types, such as lung, gastric and breast cancer, but to the best of our knowledge, its function in ESCC has not been studied in depth until the present study. It is of great significance to probe the regulatory action and underlying mechanism of miR­485­5p in ESCC. In brief, this study identified that miR­485­5p expression in ESCC tissues was significantly lower than that in normal tissues. The decrease in miR­485­5p expression was associated with a larger tumour size and poor histology and stage. The expression of miR­485­5p was relatively high in Eca 109 and TE­1 cells, but relatively low in KYSE 30. The overexpression of miR­485­5p inhibited cell proliferation, migration and invasion in vitro, whereas miR­485­5p knockdown did the opposite. Flotillin­1 (FLOT­1) can facilitate the malignant phenotype in various cancer types. The present study found that in ESCC tissue, the protein expression of FLOT­1 was negatively correlated with miR­485­5p expression. Further experiments showed that miR­485­5p directly targeted the 3'­untranslated region of FLOT­1. The overexpression of miR­485­5p significantly suppressed the mRNA and protein expression levels of FLOT­1, whereas knockdown had the reverse effects. Furthermore, overexpression of miR­485­5p restrained epithelial­mesenchymal metastasis (EMT)­related factors at both the mRNA and protein levels. At the same time, it also inhibited the growth of ESCC and restrained the EMT in vivo. In summary, miR­485­5p was found to be an inhibitor of ESCC and may have potential as a novel target candidate for ESCC treatment.

circRNA_141539 can serve as an oncogenic factor in esophageal squamous cell carcinoma by sponging miR-4469 and activating CDK3 gene.

The abnormal expression and regulation of circular RNA (circRNA) is involved in the occurrence and development of a variety of tumors. The current study aimed to determine the role of circRNA_141539 in esophageal squamous cell carcinoma (ESCC). CircRNA_141539 expression in ESCC was detected via circRNA chip analysis and verified via reverse transcription-quantitative PCR. Associations between circRNA_141539, patient clinicopathological characteristics and prognosis were also statistically analyzed. Additionally, the effects of circRNA_141539 on ESCC cell proliferation and invasion were assessed. A dual-luciferase assay was performed to analyze the interaction between circRNAs, microRNAs (miRs) and mRNAs. The results revealed that circRNA_141539 was significantly up-regulated in patients with ESCC. Furthermore, high circRNA_141539 expressions were significantly associated with TNM stage, differentiation and poor prognosis, revealing high diagnostic value (P<0.05). Furthermore, circRNA_141539 overexpression promoted cell proliferation and invasion, while circRNA_141539 silencing inhibited cell proliferation and invasion (P<0.05). The dual-luciferase reporter assay identified that circRNA_141539 directly binds to miR-4469 and also revealed that cyclin-dependent kinase-3 (CDK3) was negatively regulated by miR-4469. The results indicated that circRNA_141539 served as an oncogenic factor in ESCC by sponging miR-4469 and activating CDK3 expression. circRNA_141539 may present as a novel diagnostic and prognostic biomarker and a therapeutic target for patients with ESCC.

Retrospective analysis of the clinical effects of endoscopic mucosal dissection on treatment of early esophagogastric precancerous lesions.

INTRODUCTION: The purpose of this study was to conduct a retrospective study about the clinical effects of endoscopic mucosal dissection on the treatment of early esophagogastric precancerous lesions. METHODS: A total of 132 patients with early esophagogastric precancerous lesions who were diagnosed and treated with concurrent surgery in our hospital from January 2018 to December 2019 were included in this retrospective study. Patients were divided into endoscopic mucosal resection (EMR) group (n = 58) and endoscopic submucosal dissection (ESD) group (n = 74) according to different surgical methods. The data in the two groups were compared and analyzed in terms of surgical indicators, treatment status and incidence of postoperative complications. RESULTS: There were statistically significant differences between the two groups in the whole block cutting rate, fractional cutting rate and complete cutting rate (P < 0.05). The mean operation time of ESD group was significantly longer than that of EMR group (P < 0.05). There were no significant differences in the intraoperative bleeding rate, blood loss, average specimen area, length of hospital stay and treatment cost between the two groups (P > 0.05). The incidence and recurrence of postoperative complications, including bleeding, perforation and stenosis in the two groups, were observed within 1 year of postoperative follow-up. The incidence of complications in ESD group was slightly higher than that in EMR group, and the local recurrence rate in ESD group was lower than that in EMR group (P > 0.05). CONCLUSION: ESD is an alternative surgical treatment for patients with early esophagogastric precancerous lesions.

Does Neutrophil-to-Lymphocyte Ratio (NLR) Predict Pathologic Response to Neoadjuvant Chemoradiotherapy in Patients with Esophageal Squamous Cell Carcinoma?

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT), followed by surgery, is the current standard of care for patients with locally advanced esophageal squamous cell carcinoma. However, up to 30% of the patients do not respond to nCRT. Hence, a simple, cost-effective marker to predict response before initiation of nCRT is needed. Neutrophil-to-lymphocyte ratio (NLR) has been reported as a prognostic marker in various cancers. However, its role as a predictive marker in patients with esophageal SCC planned for nCRT has not been prospectively analyzed. MATERIALS AND METHODS: All consecutive patients with locally advanced (T1N1 and T2-T4a with or without nodal involvement) SCC planned for nCRT (CROSS protocol) followed by esophagectomy with total two field lymphadenectomy between December 2013 and December 2019 were included in this prospective analytical cohort study. NLR was calculated 1 week before starting the nCRT and was correlated with the histopathological response [Mandard tumor regression grade (TRG)]. RESULTS: Of the 216 patients with esophageal cancer evaluated during the study period, 57 patients with SCC who fulfilled the inclusion criteria were included. A good pathologic response (TRG 1 and 2) to nCRT was seen in 28 (49.1%) patients. Using a ROC curve, the optimal cutoff value of pretherapy NLR for predicting good pathologic response was 2.33. With an NLR cutoff value of 2.33, 53.3% of patients had a good pathologic response to nCRT compared with 47.6% patients with NLR ≥ 2.33 (P = 0.77). CONCLUSION: In patients with locally advanced esophageal SCC, NLR is not a useful marker to predict pathologic response to nCRT.

Downstaging and Histological Effects Might Be Reliable Predictors of the Efficacy of DOC+CDDP+5-FU (DCF) as Neoadjuvant Therapy for Stage III or Borderline Resectable Esophageal Cancer: a Single Institute Experience.

PURPOSE: In Japan, two courses of CDDP+5-FU (CF) therapy followed by surgery are accepted as a standard treatment for stage II/III esophageal cancer (EC) based on the results of the JCOG9907 trial. To gain a better survival, benefit especially for stage III patients in comparison with CF therapy, a three-arm phase III trial (neoadjuvant setting: CF vs. CF + radiation vs. DOC+CF [DCF]) is ongoing. We have aggressively performed DCF therapy for stage III or IV patients since October 2014. We herein review the outcomes of DCF therapy. METHODS: We retrospectively reviewed the cases of 27 patients with stage III or IV EC (male, n = 24; female, n = 3; median age, 70.0 years) who received DCF therapy. RESULTS: The response rate was 48.1%. Downstaging was achieved over the course of treatment in 14 patients (51.9%). Twenty-six patients transitioned to surgery, with 25 receiving R0 resection. DCF-treated patients who achieved downstaging showed significantly longer relapse-free survival (RFS) than those without downstaging (p = 0.0002). DCF-treated patients with a grade ≥ 1b histological effect showed significantly longer RFS than those with a grade < 1b effect (p = 0.0282). The multivariate analysis showed that downstaging was the only factor significantly associated with RFS in DCF-treated patients. CONCLUSIONS: DCF therapy for stage ≥ III esophageal carcinoma is both feasible and effective. These findings suggest that downstaging and the histological effect might predict the effects of DCF therapy for EC.

Role of Endoscopic Mucosal Resection and Endoscopic Submucosal Dissection in the Management of Barrett's Related Neoplasia.

Endoscopic resection has been proven to be safe and highly effective for removing early neoplastic lesions in Barrett esophagus. It enables accurate histopathological assessment and is therefore considered as the cornerstone in the endoscopic work-up for patients with Barrett neoplasia. Various techniques are available to perform endoscopic resection. Multiband mucosectomy is the most commonly used resection technique. However, endoscopic submucosal dissection is gaining ground in the Western world. Endoscopic resection for low-risk submucosal lesions already is fully justified. Future studies have to point out whether endoscopic resection and subsequent follow-up are also justified in selected patients with high-risk submucosal tumors.

Efficacy of Cryotherapy as a Primary Endoscopic Ablation Modality for Dysplastic Barrett's Esophagus and Early Esophageal Neoplasia: A Systematic Review and Meta-Analysis.

INTRODUCTION: Cryotherapy is a cold-based ablative therapy used primarily as second line therapy in patients with Barrett's esophagus (BE) who have persistent dysplasia after undergoing endoscopic treatment with radiofrequency ablation (RFA). Few studies have described the use of cryotherapy as a primary treatment modality for dysplastic or neoplastic BE. AIM: To evaluate the efficacy of cryotherapy as primary treatment of dysplastic and/or neoplastic BE by conducting a systemic review and meta-analysis. METHODS: A systematic search of Medline, Embase, and Web of Science was performed from January 2000 through March 2020. Articles included were observational studies and clinical trials which included patients who had biopsy confirmed dysplastic or neoplastic BE (i.e., high grade dysplasia (HGD), low grade dysplasia (LGD) or intramucosal adenocarcinoma (ImCA)), underwent ≥1 session of cryotherapy, and had a follow-up endoscopy. Primary outcomes were pooled proportions of patients achieving complete eradication of dysplasia (CE-D) and/or intestinal metaplasia (CE-IM) by using a random effects model. RESULTS: Fourteen studies making up 405 patients with follow-up ranging from 3-54 months were included. In 13 studies, a total of 321/405 patients achieved CE-D with a pooled proportion of 84.8% (95% confidence interval [CI] 72.2-94.4), with substantial heterogeneity (I2 = 88.3%). In 13 studies, a total of 321/405 patients achieved CE-D with a pooled proportion of 84.8% (95% confidence interval [CI] 72.2-94.4), with substantial heterogeneity (I2 = 88.3%). Subgroup analysis of only high-quality studies revealed a pooled proportion of CE-D 91.3% (95% CI, 83.0-97.4, I2 = 69.5%) and pooled proportion of CE-IM of 71.6% (95% CI, 59.0-82.9, I2 = 80.9%). Adverse events were reported in 12.2% patients. CONCLUSION: Cryotherapy is a safe and effective primary therapy for dysplastic/early neoplastic BE. CE-D and CE-IM rates are comparable to those for other ablation modalities, including RFA. Cryotherapy should be considered for primary therapy of dysplastic BE and early esophageal neoplasia.

Endoscopic mucosal resection for early esophageal carcinoma is effective and safe but necessitates continued surveillance.

BACKGROUND: Endoscopic mucosal resection (EMR) is used for the treatment of early esophageal cancer (EEC). METHODS: This a retrospective study aimed to study the efficacy, safety, and the recurrence rate of EEC following EMR. RESULTS: Seventy-nine patients who had undergone EMR for early EEC (T1a andT1b lesions) from 2006 to 2015 were included. EMR alone was considered curative in 51 patients who had T1a lesion. Complete remission was achieved in 50 (98%) patients. Mean number of sessions of EMR was 1.14. Cancer recurred locally in 6 (12%) of 50 patients at a median follow-up of 48 (18-72) months. Endoscopic treatment alone achieved complete remission at last follow up in 47 of 50 patients (94%) who had initial EMR with complete remission, or in 47 of all 51 patients (92%) in whom EMR was considered curative for EC. The Kaplan-Meier cancer-free survival following complete remission with EMR was 94.2% at 1 year and 88.4% at 5 years. Patients with complete eradication of Barrett's had lower risk of recurrence of adenocarcinoma (AC) compared with patients who had persistent Barrett's (p = 0.01). EMR alone was not considered curative in 19 patients, 16 with T1b AC and 3 with T1a squamous cell carcinoma (SCC) invading the muscularis mucosa (m3). Two major adverse events were noted: delayed bleeding requiring hospitalization, and perforation that was closed endoscopically. CONCLUSION: EMR is effective and safe for the management of early EC. The risk of cancer recurrence, albeit small, warrants surveillance. Complete eradication of Barrett's should be attempted in all patients after EMR of AC.

Multifocal Cryoballoon Ablation for Eradication of Barrett's Esophagus-Related Neoplasia: A Prospective Multicenter Clinical Trial.

INTRODUCTION: Ablation of Barrett's esophagus (BE) is the preferred approach for the treatment of neoplasia without visible lesions. Limited data on cryoballoon ablation (CBA) suggest its potential clinical utility. We evaluated the safety and efficacy of CBA in a multicenter study of patients with neoplastic BE. METHODS: In a prospective clinical trial, 11 academic and community centers recruited consecutive patients with BE of 1-6 cm length and low-grade dysplasia, high-grade dysplasia (HGD), or intramucosal adenocarcinoma (ImCA) confirmed by central pathology. Patients with symptomatic pre-existing strictures or visible BE lesions had dilation or endoscopic mucosal resection (EMR), respectively, before enrollment. A nitrous oxide cryoballoon focal ablation system was used to treat all visible columnar mucosa in up to 5 sessions. Study end points included complete eradication of all dysplasia (CE-D) and intestinal metaplasia (CE-IM) at 1 year. RESULTS: One hundred twenty patients with BE with ImCA (20%), HGD (56%), or low-grade dysplasia (23%) were enrolled. In the intention-to-treat analysis, the CE-D and CE-IM rates were 76% and 72%, respectively. In the per-protocol analysis (94 patients), the CE-D and CE-IM rates were 97% and 91%, respectively. Postablation pain was mild and short lived. Fifteen subjects (12.5%) developed strictures requiring dilation. One patient (0.8%) with HGD progressed to ImCA, which was successfully treated with EMR. Another patient (0.8%) developed gastrointestinal bleeding associated with clopidogrel use. One patient (0.8%) had buried BE with HGD in 1 biopsy, not confirmed by subsequent EMR. DISCUSSION: In patients with neoplastic BE, CBA was safe and effective. Head-to-head comparisons between CBA and other ablation modalities are warranted (clinicaltrials.gov registration NCT02514525).

Submucosal Tunneling Endoscopic Resection.

Minimally invasive endoscopic resection procedures continue to evolve, with submucosal tunneling endoscopic resection (STER) being a durable option for en bloc resection of submucosal tumors. Whether STER can be effectively used for larger (>3.5 cm) lesions remains to be seen. STER-ET is a novel approach for removal of extraluminal tumors, but data are currently limited to support its use.

Per Oral Zenker Diverticulotomy.

Zenker diverticulum (ZD) is a rare but treatable surgical disease affecting the elderly. This article reviews current available evidence and management of ZD, which includes open surgical, rigid endoscopic, and flexible endoscopic diverticulotomy with common goal of complete division of cricopharyngeus muscle. Careful patient selection and operative intervention tailored to patient characteristics is important when evaluating patients for operative intervention for ZD. Described in detail is a novel flexible endoscopic approach using submucosal tunneling technique to perform cricopharyngeal myotomy, also called per oral endoscopic myotomy, which is demonstrated to be safe and effective in the management of ZD with low morbidity.

Paraneoplastic leukemoid reaction in a localised squamous cell oesophageal cancer with paracrine G-CSF production.

A 51-year-old-man presented with symptoms and baseline investigations suggestive of an infective process. Most strikingly, there was a pronounced neutrophil predominant leucocytosis. Lack of a clinical and biochemical response to empirical antibiotic therapy, prompted imaging for a deep-seated infective process, incidentally uncovering a gastro-oesophageal junction tumour. Resection of the tumour was followed by rapid resolution of the leucocytosis. He remains in clinical remission since tumour resection and adjuvant chemotherapy. Cancer-associated leukemoid reactions in non-disseminated tumours are rare. The role of polymorphonuclear (PMN) leucocytes both in the peripheral blood and the tumour itself is discussed herein. There is increasing recognition of the importance of the non-cancer cellular components of the tumour microenvironment. Myeloid suppressor cells are a subset of PMN leucocytes which play a role in tumour progression.The role of these cells and granulocyte colony-stimulating factor is highlighted in this case.

Targeted Sequencing of Sorted Esophageal Adenocarcinoma Cells Unveils Known and Novel Mutations in the Separated Subpopulations.

INTRODUCTION: Our study aimed at investigating tumor heterogeneity in esophageal adenocarcinoma (EAC) cells regarding clinical outcomes. METHODS: Thirty-eight surgical EAC cases who underwent gastroesophageal resection with lymph node dissection in 3 university centers were included. Archival material was analyzed via high-throughput cell sorting technology and targeted sequencing of 63 cancer-related genes. Low-pass sequencing and immunohistochemistry (IHC) were used to validate the results. RESULTS: Thirty-five of 38 EACs carried at least one somatic mutation that was absent in the stromal cells; 73.7%, 10.5%, and 10.5% carried mutations in tumor protein 53, cyclin dependent kinase inhibitor 2A, and SMAD family member 4, respectively. In addition, 2 novel mutations were found for hepatocyte nuclear factor-1 alpha in 2 of 38 cases. Tumor protein 53 gene abnormalities were more informative than p53 IHC. Conversely, loss of SMAD4 was more frequently noted with IHC (53%) and was associated with a higher recurrence rate (P = 0.015). Only through cell sorting we were able to detect the presence of hyperdiploid and pseudodiploid subclones in 7 EACs that exhibited different mutational loads and/or additional copy number amplifications, indicating the high genetic heterogeneity of these cancers. DISCUSSION: Selective cell sorting allowed the characterization of multiple molecular defects in EAC subclones that were missed in a significant number of cases when whole-tumor samples were analyzed. Therefore, this approach can reveal subtle differences in cancer cell subpopulations. Future studies are required to investigate whether these subclones are responsible for treatment response and disease recurrence.

Repeated Endoscopic Submucosal Dissection for Esophageal Neoplasia Located Close to a Previous Endoscopic Submucosal Dissection Scar.

INTRODUCTION: Endoscopic submucosal dissection (ESD) could become a standard treatment for early stage esophageal neoplasia. Recurrence sometimes develops close to a previous ESD scar. These lesions are predictably difficult to treat with ESD because of severe fibrosis. We evaluated the clinical outcomes of ESD for esophageal neoplasia located close to a previous ESD scar. METHODS: This was a retrospective observational study in a single institution. A total of 549 consecutive patients with 927 esophageal lesions were treated with ESD. The primary outcomes were resectability and adverse events of esophageal neoplasia located close to previous ESD scars (ESD scar group) than in primary esophageal ESD (primary group). Furthermore, predictive factors of perforation were examined. RESULTS: A total of 545 primary and 29 ESD scars in consecutive patients were evaluated. En bloc and complete (R0) resection rates in the ESD scar group were lower than those in the primary group (79.3% vs 98.3%, P < 0.01 and 75.9% vs 93.4%, P < 0.01). Perforations occurred more frequently in the ESD scar group (10.3% vs 2.0%, P = 0.03). The ESD scar group was a predictive factor for perforation (odds ratio = 10.37, 95% confidence interval: 2.15-49.94, P = 0.004). There were similar results for inverse probability of treatment weighting methods (odds ratio = 6.78, 95% confidence interval: 1.40-32.98, P = 0.018). DISCUSSION: ESD for esophageal neoplasia located close to a previous ESD scar was difficult to completely resect and increased the likelihood of perforation but could be a treatment option.

Outcomes of Hemospray therapy in the treatment of intraprocedural upper gastrointestinal bleeding post-endoscopic therapy.

INTRODUCTION: With increasing advances in minimally invasive endoscopic therapies and endoscopic resection techniques for luminal disease, there is an increased risk of post-procedure bleeding. This can contribute to significant burden on patient's quality of life and health resources when reintervention is required. Hemospray (Cook Medical, North Carolina, USA) is a novel haemostatic powder licensed for gastrointestinal bleeding. The aim of this single-arm, prospective, non-randomised multicentre international study is to look at outcomes in patients with upper gastrointestinal bleeds following elective endoscopic therapy treated with Hemospray to achieve haemostasis. METHODS: Data was prospectively collected on the use of Hemospray from 16 centres (January 2016-November 2019). Hemospray was used during the presence of progressive intraprocedural bleeding post-endoscopic therapy as a monotherapy, dual therapy with standard haemostatic techniques or rescue therapy once standard methods had failed. Haemostasis was defined as the cessation of bleeding within 5 min of the application of Hemospray. Re-bleeding was defined as a sustained drop in haemoglobin (>2 g/l), haematemesis or melaena with haemodynamic instability after the index endoscopy. RESULTS: A total of 73 patients were analysed with bleeding post-endoscopic therapy. The median Blatchford score at baseline was five (interquartile range 0-9). The median Rockall score was six (interquartile range 5-7). Immediate haemostasis following the application of Hemospray was achieved in 73/73 (100%) of patients. Two out of 57 (4%) had a re-bleed post-Hemospray, one was following oesophageal endoscopic mucosal resection and the other post-duodenal endoscopic mucosal resection. Both patients had a repeat endoscopy and therapy within 24 h. Re-bleeding data was missing for 16 patients, and mortality data was missing for 14 patients. There were no adverse events recorded in association with the use of Hemospray. CONCLUSION: Hemospray is safe and effective in achieving immediate haemostasis following uncontrolled and progressive intraprocedural blood loss post-endoscopic therapy, with a low re-bleed rate.