Outcomes of Heller Myotomy for Esophageal Achalasia: Lessons From a 48-Year Prospective Experience With 4 Different Techniques.

OBJECTIVE: To provide information on long-term outcomes of Heller myotomy for esophageal achalasia with or without an antireflux fundoplication. BACKGROUND: Since the adoption of the Heller myotomy, surgeons have modified the original technique in order to balance the cure of dysphagia and the consequent cardial incontinence. METHODS: Totally, 470 patients underwent primary Heller myotomy between 1955 and 2020. A long abdominal myotomy (AM) was performed in 83 patients, the Ellis limited transthoracic myotomy (TM) in 30, the laparotomic Heller-Dor (L-HD) in 202, the videolaparoscopic Heller-Dor (VL-HD) in 155. The HD was performed under intraoperative manometric assessment. Starting on 1973 these patients underwent a prospective follow-up program of timed lifelong clinical, radiological, endoscopic evaluations. RESULTS: Median follow-up time was 23.06 years [interquantile range (IQR): 15.04-32.06] for AM, 29.22 years (IQR: 13.46-40.17) for TM, 14.85 years (IQR: 11.05-21.56) for L-HD and 7.51 years (IQR: 3.25-9.60) for VL-HD. In AM, relapse of dysphagia occurred in 25/71 (35.21%), in TM in 11/30 (36.66%), in LH-D in 10/201 (4.97%), in VL-HD in 3/155 (1.93%). Erosive-ulcerative esophagitis was diagnosed for AM in 28.16%, for TM in 30%, for L-HD in 8.45%, for VL-HD in 2.58%. Overall, the outcome was satisfactory in 52.11% for AM, 41.9% for TM, 89.05% for L-HD, 96.12% for VL-HD. CONCLUSIONS: The Dor fundoplication drastically reduces postmyotomy gastroesophageal reflux. The Heller-Dor operation is a competitive option for the cure of esophageal achalasia if this operation is performed according to the rules of surgical physiology learned by means of intraoperative manometry.

True Short Esophagus in Gastroesophageal Reflux Disease: Old Controversies With New Perspectives.

OBJECTIVE: To explore the true short esophagus (TSE) frequency and long-term results of patients undergoing gastroesophageal reflux disease (GERD) or hiatus hernia (HH) surgery. BACKGROUND: The existence and treatment of TSE during GERD/HH surgery is controversial. Satisfactory long-term results have been achieved with and without surgical techniques dedicated to TSE. METHODS: In 311 consecutive patients undergoing minimally invasive surgery for GERD/HH, the distance between the endoscopically-localized gastroesophageal junction (GEJ) and the apex of the diaphragmatic hiatus after maximal thoracic esophagus mobilization was measured. A standard Nissen fundoplication (SN) was performed in cases with an abdominal length >1.5 cm; in cases of TSE (abdominal length <1.5 cm), a Collis-Nissen (CN) or stomach around the stomach fundoplication (SASF) in elderly patients was performed. The fundoplication superior margin was fixed below the hiatus, but over the GEJ. The patients' symptoms, and radiological and endoscopic data were pre/postoperatively recorded. RESULTS: After intrathoracic esophageal mobilization (median 9 cm), TSE was diagnosed in 31.8% of 311 cases. With a median follow-up of 96 months (309 patients), HH relapse was radiologically diagnosed in 3.2% of patients, with excellent, good, fair, and poor outcomes in 45.6%, 44.3%, 6.2%, and 3.9% of cases, respectively, and no significant differences among SN (68.5%), CN (26.4%), and SASF (5.2%). CONCLUSIONS: TSE was present in 31.8% of patients routinely submitted to GERD/HH surgery. In the presence of TSE, CN and SASF performed according to determined surgical principles may achieve similar satisfactory results. This finding warrants confirmation with a prospective multicenter study.

Risk of Squamous Cell Carcinoma and Adenocarcinoma of the Esophagus in Patients With Achalasia: A Long-Term Prospective Cohort Study in Italy.

INTRODUCTION: Epidemiological studies assessing relative risk and incidence rate of esophageal cancer in patients with achalasia are scarce. We performed a long-term prospective cohort study to evaluate the risk of both squamous cell carcinoma and adenocarcinoma of the esophagus in these patients. METHODS: Between 1973 and 2018, patients with primary achalasia were followed by the same protocol including upper endoscopy with esophageal biopsies. Standardized incidence ratios (SIRs) with 95% confidence interval (CI) were used to estimate the relative risk of esophageal cancer in patients with achalasia compared with the sex- and age-matched general population. RESULTS: A cohort of 566 patients with achalasia (46% men, mean age at diagnosis: 48.1 years) was followed for a mean of 15.5 years since the diagnosis of achalasia. Overall, 20 patients (15 men) developed esophageal cancer: 15 squamous cell carcinoma and 5 adenocarcinoma. The risk of esophageal cancer was significantly greater than the general population (SIR 104.2, 95% CI 63.7-161), and this for both squamous cell carcinoma (SIR 126.9, 95% CI 71.0-209.3) and adenocarcinoma (SIR 110.2, 95% CI 35.8-257.2). The excess risk was higher in men than women. Annual incidence rate of esophageal cancer was only 0.24% and was higher for squamous cell carcinoma (0.18%) than adenocarcinoma (0.06%). DISCUSSION: Patients with achalasia have an excess risk of developing both squamous cell carcinoma and adenocarcinoma of the esophagus; however, this prospective cohort study confirms that the annual incidence of esophageal cancer is rather low. These findings may have implications for endoscopic surveillance of patients with achalasia.

Exploring the concept of centralization of surgery for benign esophageal diseases: a Delphi based consensus from the European Society for Diseases of the Esophagus.

Surgery for benign esophageal diseases may be complex, requiring specialist training, but currently, unlike oncologic surgery, it is not centralized. The aim of the study was to explore the opinion of European surgeons on the centralization of surgery for benign esophageal diseases. A web-based questionnaire, developed through a modified Delphi process, was administered to general and thoracic surgeons of 33 European surgical societies. There were 791 complete responses (98.5%), in 59.2% of respondents, the age ranged between 41 and 60 years, 60.3% of respondents worked in tertiary centers. In 2017, the number of major surgical procedures performed for any esophageal disease by respondents was <10 for 56.5% and >100 for 4.5%; in responder's hospitals procedures number was <10 in 27% and >100 in 15%. Centralization of surgery for benign esophageal diseases was advocated by 83.4%, in centers located according to geographic/population criteria (69.3%), in tertiary hospitals (74.5%), with availability of advanced diagnostic and interventional technologies (88.4%), in at least 10 beds units (70.5%). For national and international centers accreditation/certification, criteria approved included in-hospital mortality and morbidity (95%), quality of life oriented follow-up after surgery (88.9%), quality audits (82.6%), academic research (58.2%), and collaboration with national and international centers (76.6%); indications on surgical procedures volumes were variable. The present study strongly supports the centralization of surgery for benign esophageal diseases, in large part modeled on the principles that have underpinned the centralization of cancer surgery internationally, with emphasis on structure, process, volumes, quality audit, and clinical research.

How to select patients for antireflux surgery? The ICARUS guidelines (international consensus regarding preoperative examinations and clinical characteristics assessment to select adult patients for antireflux surgery).

OBJECTIVE: Antireflux surgery can be proposed in patients with GORD, especially when proton pump inhibitor (PPI) use leads to incomplete symptom improvement. However, to date, international consensus guidelines on the clinical criteria and additional technical examinations used in patient selection for antireflux surgery are lacking. We aimed at generating key recommendations in the selection of patients for antireflux surgery. DESIGN: We included 35 international experts (gastroenterologists, surgeons and physiologists) in a Delphi process and developed 37 statements that were revised by the Consensus Group, to start the Delphi process. Three voting rounds followed where each statement was presented with the evidence summary. The panel indicated the degree of agreement for the statement. When 80% of the Consensus Group agreed (A+/A) with a statement, this was defined as consensus. All votes were mutually anonymous. RESULTS: Patients with heartburn with a satisfactory response to PPIs, patients with a hiatal hernia (HH), patients with oesophagitis Los Angeles (LA) grade B or higher and patients with Barrett's oesophagus are good candidates for antireflux surgery. An endoscopy prior to antireflux surgery is mandatory and a barium swallow should be performed in patients with suspicion of a HH or short oesophagus. Oesophageal manometry is mandatory to rule out major motility disorders. Finally, oesophageal pH (±impedance) monitoring of PPI is mandatory to select patients for antireflux surgery, if endoscopy is negative for unequivocal reflux oesophagitis. CONCLUSION: With the ICARUS guidelines, we generated key recommendations for selection of patients for antireflux surgery.

Distribution of lymph node metastases in esophageal carcinoma [TIGER study]: study protocol of a multinational observational study.

BACKGROUND: An important parameter for survival in patients with esophageal carcinoma is lymph node status. The distribution of lymph node metastases depends on tumor characteristics such as tumor location, histology, invasion depth, and on neoadjuvant treatment. The exact distribution is unknown. Neoadjuvant treatment and surgical strategy depends on the distribution pattern of nodal metastases but consensus on the extent of lymphadenectomy has not been reached. The aim of this study is to determine the distribution of lymph node metastases in patients with resectable esophageal or gastro-esophageal junction carcinoma in whom a transthoracic esophagectomy with a 2- or 3-field lymphadenectomy is performed. This can be the foundation for a uniform worldwide staging system and establishment of the optimal surgical strategy for esophageal cancer patients. METHODS: The TIGER study is an international observational cohort study with 50 participating centers. Patients with a resectable esophageal or gastro-esophageal junction carcinoma in whom a transthoracic esophagectomy with a 2- or 3-field lymphadenectomy is performed in participating centers will be included. All lymph node stations will be excised and separately individually analyzed by pathological examination. The aim is to include 5000 patients. The primary endpoint is the distribution of lymph node metastases in esophageal and esophago-gastric junction carcinoma specimens following transthoracic esophagectomy with at least 2-field lymphadenectomy in relation to tumor histology, tumor location, invasion depth, number of lymph nodes and lymph node metastases, pre-operative diagnostics, neo-adjuvant therapy and (disease free) survival. DISCUSSION: The TIGER study will provide a roadmap of the location of lymph node metastases in relation to tumor histology, tumor location, invasion depth, number of lymph nodes and lymph node metastases, pre-operative diagnostics, neo-adjuvant therapy and survival. Patient-tailored treatment can be developed based on these results, such as the optimal radiation field and extent of lymphadenectomy based on the primary tumor characteristics. TRIAL REGISTRATION: NCT03222895 , date of registration: July 19th, 2017.

Genomic profiles of primary and metastatic esophageal adenocarcinoma identified via digital sorting of pure cell populations: results from a case report.

BACKGROUND: We report on a female patient who underwent primary radical resection for a stage 2B Her-2-positive Barrett's-type esophageal adenocarcinoma (EAC). Despite Her-2 targeted therapy, her disease recurred and required repeated metastectomies. CASE PRESENTATION: Digital cell sorting and targeted sequencing of cancer sub-clones from EAC and metastases revealed a completely mutated TP53, whereas the sorted stromal cells were wild-type. Her-2 amplification was significantly lower in the metastases when the patient became therapy-resistant. CONCLUSIONS: The mechanism of therapy resistance illustrated by this case could only be detected through accurate analysis of tumor sub-populations. Investigating tumor sub-populations of recurrent disease is important for adjusting therapy in recurrent EAC.

Ex vivo pulmonary nodule detection with miniaturized ultrasound convex probes.

BACKGROUND: The intraoperative localization of small and deep pulmonary nodules is often difficult during minimally invasive thoracic surgery. We compared the performance of three miniaturized ultrasound (US) convex probes, one of which is currently used for thoracic endoscopic diagnostic procedures, for the detection of lung nodules in an ex vivo lung perfusion model. METHODS: Three porcine cardiopulmonary blocks were perfused, preserved at 4°C for 6 h and reconditioned. Lungs were randomly seeded with different patterns of echogenicity target nodules (9 water balls, 10 fat, and 11 muscles; total n = 30). Three micro-convex US probes were assessed in an open setting on the pleural surface: PROBE 1, endobronchial US 5-10 MHz; PROBE 2, laparoscopic 4-13 MHz; PROBE 3, fingertip micro-convex probe 5-10 MHz. US probes were evaluated regarding the number of nodules localized/not localized, the correlation between US and open specimen measurements, and imaging quality. RESULTS: For detecting target nodules, the sensitivity was 100% for PROBE 1, 86.6% for PROBE 2, and 78.1% for PROBE 3. A closer correlation between US and open specimen measurements of target diameter (r = 0.87; P = 0.0001) and intrapulmonary depth (r = 0.97; P = 0.0001) was calculated for PROBE 1 than for PROBES 2 and 3. The imaging quality was significantly higher for PROBE 1 than for PROBES 2 and 3 (P < 0.04). CONCLUSIONS: US examination with micro-convex probes to detect pulmonary nodules is feasible in an ex vivo lung perfusion model. PROBE 1 achieved the best performance. Clinical research with the endobronchial US micro-convex probe during minimally invasive thoracic surgery is advisable.

Sublobar resection versus lobectomy for stage I non-small cell lung cancer: an appropriate choice in elderly patients?

PURPOSES: The aim of this study was to evaluate whether sublobar resection could achieve recurrence and survival rates equivalent to lobectomy in high-risk elderly patients. METHODS: We conducted a retrospective multicenter study that including all consecutive patients (aged >75 years) who underwent operation for clinical stage I non-small cell lung cancer (NSCLC). The clinicopathological data, postoperative morbidity and mortality, recurrence rate and vital status were retrieved. The overall survival, cancer-specific survival and disease-free survival were also assessed. RESULTS: Two hundred and thirty-nine patients (median age 78 years) were enrolled. Lobectomies were performed in 149 (62.3 %) patients and sublobar resections in 90 (39 segmentectomies, 51 wedge resections). There were no differences in the recurrence rates following lobar versus sublobar resections (19 versus 23 %, respectively; p = 0.5) or the overall survival (p = 0.1), cancer-specific survival (p = 0.3) or disease-free survival (p = 0.1). After adjusting for 1:1 propensity score matching and a matched pair analysis, the results remained unchanged. A tumor size >2 cm and pN2 disease were independent negative prognostic factors in unmatched (p = 0.01 and p = 0.0003, respectively) and matched (p = 0.02 and p = 0.005, respectively) analyses. CONCLUSIONS: High-risk elderly patients may benefit from sublobar resection, which provides an equivalent long-term survival compared to lobectomy.

Effectiveness of antireflux surgery for the cure of chronic cough associated with gastroesophageal reflux disease.

BACKGROUND: The effectiveness of surgical therapy for chronic cough secondary to gastroesophageal reflux disease remains controversial. The purpose of this study was to assess the efficacy of surgery and to identify the preoperative clinical profile that could predict the positive effects of treatment on chronic cough. STUDY DESIGN: Of 299 patients who underwent antireflux surgery between 1995 and 2010, 67 patients were affected by chronic cough and typical symptoms. In addition, 83 patients with typical symptoms were selected to form the control group, according to the parameters of age, sex, and the period of surgical activity. Preoperatively, all patients underwent a workup, including symptom assessment, barium swallow, upper gastrointestinal endoscopy, esophageal manometry, and 24-h pH recording or intraluminal impedance/pH monitoring in the absence of esophagitis. Patients with chronic cough also were administered a high-resolution computed tomography scan of the chest, a methacholine challenge test, and spirometry. Surgery was performed on patients positive for gastroesophageal reflux disease and negative for pulmonary diseases. The patients were followed up for a median of 84 months after surgery. RESULTS: No significant differences in preoperative reflux symptoms or esophagitis were found between the two groups. After surgery, chronic cough was absent in 57 (85 %) patients. Of the ten patients who still reported chronic cough, reflux symptoms relapsed in five, two of whom developed esophagitis. In the other five patients, typical symptoms were absent, and their chronic cough had improved but had not disappeared. CONCLUSIONS: Surgery is effective for the treatment of chronic cough secondary to gastroesophageal reflux disease, particularly if associated with severe and long-standing typical symptoms.

miRNA-221 and miRNA-483-3p Dysregulation in Esophageal Adenocarcinoma.

Alterations in microRNA (miRNA) expression have been reported in different cancers. We assessed the expression of 754 oncology-related miRNAs in esophageal adenocarcinoma (EAC) samples and evaluated their correlations with clinical parameters. We found that miR-221 and 483-3p were consistently upregulated in EAC patients vs. controls (Wilcoxon signed-rank test: miR-221 p < 0.0001; miR-483-3p p < 0.0001). Kaplan-Meier analysis showed worse cancer-related survival among all EAC patients expressing high miR-221 or miR-483-3p levels (log-rank p = 0.0025 and p = 0.0235, respectively). Higher miR-221 or miR-483-3p levels also correlated with advanced tumor stages (Mann-Whitney p = 0.0195 and p = 0.0085, respectively), and overexpression of miR-221 was associated with worse survival in low-risk EAC patients. Moreover, a significantly worse outcome was associated with the combined overexpression of miR-221 and miR-483-3p (log-rank p = 0.0410). To identify target genes affected by miRNA overexpression, we transfected the corresponding mimic RNA (miRVANA) for either miR-221 or miR-483-3p in a well-characterized esophageal adenocarcinoma cell line (OE19) and performed RNA-seq analysis. In the miRNA-overexpressing cells, we discovered a convergent dysregulation of genes linked to apoptosis, ATP synthesis, angiogenesis, and cancer progression, including a long non-coding RNA associated with oncogenesis, i.e., MALAT1. In conclusion, dysregulated miRNA expression, especially overexpression of miR-221 and 483-3p, was found in EAC samples. These alterations were connected with a lower cancer-specific patient survival, suggesting that these miRNAs could be useful for patient stratification and prognosis.

Detection of a Novel MSI2-C17orf64 Transcript in a Patient with Aggressive Adenocarcinoma of the Gastroesophageal Junction: A Case Report.

Adenocarcinoma of the esophagus (EAC) and gastroesophageal junction (GEJ-AC) is associated with poor prognosis, treatment resistance and limited systemic therapeutic options. To deeply understand the genomic landscape of this cancer type, and potentially identify a therapeutic target in a neoadjuvant chemotherapy non-responder 48-year-old man, we adopted a multi-omic approach. We simultaneously evaluated gene rearrangements, mutations, copy number status, microsatellite instability and tumor mutation burden. The patient displayed pathogenic mutations of the TP53 and ATM genes and variants of uncertain significance of three kinases genes (ERBB3, CSNK1A1 and RPS6KB2), along with FGFR2 and KRAS high copy number amplification. Interestingly, transcriptomic analysis revealed the Musashi-2 (MSI2)-C17orf64 fusion that has never been reported before. Rearrangements of the RNA-binding protein MSI2 with a number of partner genes have been described across solid and hematological tumors. MSI2 regulates several biological processes involved in cancer initiation, development and resistance to treatment, and deserves further investigation as a potential therapeutic target. In conclusion, our extensive genomic characterization of a gastroesophageal tumor refractory to all therapeutic approaches led to the discovery of the MSI2-C17orf64 fusion. The results underlie the importance of deep molecular analyses enabling the identification of novel patient-specific markers to be monitored during therapy or even targeted at disease evolution.

Correlations between Molecular Alterations, Histopathological Characteristics, and Poor Prognosis in Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a severe malignancy with increasing incidence, poorly understood pathogenesis, and low survival rates. We sequenced 164 EAC samples of naïve patients (without chemo-radiotherapy) with high coverage using next-generation sequencing technologies. A total of 337 variants were identified across the whole cohort, with TP53 as the most frequently altered gene (67.27%). Missense mutations in TP53 correlated with worse cancer-specific survival (log-rank p = 0.001). In seven cases, we found disruptive mutations in HNF1alpha associated with other gene alterations. Moreover, we detected gene fusions through massive parallel sequencing of RNA, indicating that it is not a rare event in EAC. In conclusion, we report that a specific type of TP53 mutation (missense changes) negatively affected cancer-specific survival in EAC. HNF1alpha was identified as a new EAC-mutated gene.

Quality of Life After Operation for End-Stage Achalasia: Pull-Down Heller-Dor Versus Esophagectomy.

BACKGROUND: Therapy for end-stage achalasia is debated, and data on long-term functional results of myotomy and esophagectomy are lacking. We compared quality of life and objective outcomes after pull-down Heller-Dor and esophagectomy. METHODS: The study included 32 patients, aged 57 years (interquartile range [IQR], 49-70 years), who underwent the Heller-Dor operation with verticalization of the distal esophagus in case of first instance treatment or failed surgery caused by insufficient myotomy, and 16 patients, aged 58 years (IQR, 49-67 years; P = .806), who underwent esophagectomy after failed surgery for other causes. Data were extracted from a database designed for prospective clinical research. Postoperative dysphagia, reflux symptoms, and endoscopic esophagitis were graded by semiquantitative scales. Quality of life was assessed with the 36-Item Short Form Health Survey questionnaire. RESULTS: The median follow-up period was 68 months (IQR, 40.43-94.48 months) after pull-down Heller-Dor and 61 months (IQR 43.72-181.43 months) after esophagectomy (P = .598). No statistically significant differences were observed for dysphagia (P = .948), reflux symptoms (P = .186), or esophagitis (P = .253). No statistically significant differences were observed in the domains physical functioning (P = .092), bodily pain (P = .075) or general health (P = .453). Significant differences were observed in favor of pull-down Heller-Dor for the domains role physical (100 vs 100, P = .043), role emotional (100 vs 0, P = .002), vitality (90 vs 55, P< .001), mental health (92 vs 68, P = .002), and social functioning (100 v s75, P = .011). CONCLUSIONS: The pull-down Heller-Dor achieved objective results similar to those of esophagectomy with a better quality of life. This technique may be the first choice for end-stage achalasia in patients with null or low risk for cancer or after recurrent dysphagia caused by insufficient myotomy.

Sarcomatoid change in adenocarcinoma arising in adulthood congenital pulmonary airway malformation.

Congenital pulmonary airway malformations (CPAM) are rare conditions generally diagnosed in childhood and possibly harboring malignant tumor growths. We describe a unique case of pleomorphic carcinoma in a longstanding type 1 CPAM diagnosed by wedge resection. The patient underwent completion left lower lobectomy and lymphadenectomy, but cancer recurred in nodal station #7 six months later. Clinicians should keep in mind that CPAM may hide radiologically undetectable malignancy in a relevant rate of cases, then requiring surgery in all patients. While MIA is the most common histology in type 1 CPAM, sarcomatoid change has herein been demonstrated.

Prognostic Significance of Pulmonary Multifocal Neuroendocrine Proliferation With Typical Carcinoid.

BACKGROUND: The clinical significance of multifocal pulmonary neuroendocrine proliferation (MNEP), including tumorlets and pulmonary neuroendocrine cell hyperplasia, in association with typical carcinoid (TC), is still debated. METHODS: We evaluated a retrospective series of TC with long-term follow-up data prospectively collected from 2 institutions and compared the outcome between TC alone and MNEP plus TC. Several baseline covariates were imbalanced between the MNEP plus TC and TC groups; therefore, we conducted 1:1 propensity score matching and inverse probability of treatment weighting in the full sample. In the matched group, the association of clinical, respiratory, and work-related factors with the group was determined through univariable and multivariable conditional logistic regression analysis. RESULTS: A total of 234 TC patients underwent surgery: 41 MNEP plus TC (17.5%) and 193 TC alone (82.5%). In the MNEP plus TC group, older age (P < .001), peripheral tumors (P = .0032), smaller tumor size (P = .011), and lymph node spread (P = .02) were observed compared with the TC group. Relapses occurred in 8 patients in the MNEP plus TC group (19.5%) and 7 in the TC group (3.6%). After matching, in 36 pairs of patients, a significantly higher 5-year progression-free rate was observed for the TC group (P < .01). Similar results were observed using inverse probability of treatment weighting in the full sample. The odds of being in the MNEP plus TC group was higher for those with work-related exposure to inhalant agents (P = .008), asthma or bronchitis (P = .002), emphysema, fibrosis, and inflammatory status (P = .032), or micronodules on the chest computed tomography scan and respiratory insufficiency (P = .036). CONCLUSIONS: The association with MNEP seems to represent a clinically and prognostic relevant factor in TC. Hence, careful preoperative workup, systematic pathologic evaluation, including nontumorous lung parenchyma, and long-term postoperative follow-up should be recommended in these patients.

Selective targeting BMP2 and 4 in SMAD4 negative esophageal adenocarcinoma inhibits tumor growth and aggressiveness in preclinical models.

PURPOSE: Abnormalities within the Sonic Hedgehog (SHH), Bone Morphogenetic Protein (BMP) and SMAD4 signalling pathways have been associated with the malignant behavior of esophageal adenocarcinoma (EAC). We recently developed two specific llama-derived antibodies (VHHs), C4C4 and C8C8, which target BMP4 and BMP2/4, respectively. Here we aimed to demonstrate the feasibility of the VHHs for the treatment of EAC and to elucidate its underlying mechanism. METHODS: Gene Set Enrichment Analysis (GSEA) was performed on a TCGA dataset, while expression of SHH, BMP2/4 and SMAD4 was validated in a cohort of EAC patients. The effects of the VHHs were tested on the recently established SMAD4(-) ISO76A primary EAC cell line and its counterpart SMAD4(+) ISO76A. In a patient-derived xenograft (PDX) model, the VHHs were evaluated for their ability to selectively target tumor cells and for their effects on tumor growth and survival. RESULTS: High expression of BMP2/4 was detected in all SMAD4 negative EACs. SHH upregulated BMP2/4 expression and induced p38 MAPK signaling in the SMAD4(-) ISO76A cells. Inhibition of BMP2/4 by VHHs decreased the aggressive and chemo-resistant phenotype of the SMAD4(-) ISO76A but not of the SMAD4(+) ISO76A cells. In the PDX model, in vivo imaging indicated that VHHs effectively targeted tumor cells. Both VHHs significantly inhibited tumor growth and acted synergistically with cisplatin. Furthermore, we found that C8C8 significantly improved survival of the mice. CONCLUSIONS: Our data indicate that increased BMP2/4 expression triggers aggressive non-canonical BMP signaling in SMAD4 negative EAC. Inhibiting BMP2/4 decreases malignant behavior and improves survival. Therefore, VHHs directed against BMP2/4 hold promise for the treatment of SMAD4 negative EAC.

Identification of Novel Molecular Subgroups in Esophageal Adenocarcinoma to Predict Response to Neo-Adjuvant Therapies.

Esophageal adenocarcinoma (EAC) is a highly aggressive cancer and its response to chemo- and radiotherapy is unpredictable. EACs are highly heterogeneous at the molecular level. The aim of this study was to perform gene expression analysis of EACs to identify distinct molecular subgroups and to investigate expression signatures in relation to treatment response. In this prospective observational study, RNA sequencing was performed on pre-treatment endoscopic EAC biopsies from a discovery cohort included between 2012 and 2017 in one Dutch Academic Center. Four additional cohorts were analyzed for validation purposes. Unsupervised clustering was performed on 107 patients to identify biological EAC subgroups. Specific cell signaling profiles were identified and evaluated with respect to predicting response to neo-adjuvant chemo(radio)therapy. We identified and validated three distinct biological EAC subgroups, characterized by (1) p38 MAPK/Toll-like receptor signaling; (2) activated immune system; and (3) impaired cell adhesion. Subgroup 1 was associated with poor response to chemo-radiotherapy. Moreover, an immune signature with activated T-cell signaling, and increased number of activated CD4 T memory cells, neutrophils and dendritic cells, and decreased M1 and M2 macrophages and plasma cells, was associated with complete histopathological response. This study provides a novel molecular classification for EACs. EAC subgroup 1 proved to be more therapy-resistant, while immune signaling was increased in patients with complete response to chemo-radiotherapy. Our findings give insight into the biology of EACs and in cellular signaling mechanisms underlying response to neo-adjuvant treatment. Future implementation of this classification will improve patient stratification and enhance the development of targeted therapies.

The Prognostic Impact of Histology in Esophageal and Esophago-Gastric Junction Adenocarcinoma.

Stage significantly affects survival of esophageal and esophago-gastric junction adenocarcinomas (EA/EGJAs), however, limited evidence for the prognostic role of histologic subtypes is available. The aim of the study was to describe a morphologic approach to EA/EGJAs and assess its discriminating prognostic power. Histologic slides from 299 neoadjuvant treatment-naïve EA/EGJAs, resected in five European Centers, were retrospectively reviewed. Morphologic features were re-assessed and correlated with survival. In glandular adenocarcinomas (240/299 cases-80%), WHO grade and tumors with a poorly differentiated component ≥6% were the most discriminant factors for survival (both p < 0.0001), distinguishing glandular well-differentiated from poorly differentiated adenocarcinomas. Two prognostically different histologic groups were identified: the lower risk group, comprising glandular well-differentiated (34.4%) and rare variants, such as mucinous muconodular carcinoma (2.7%) and diffuse desmoplastic carcinoma (1.7%), versus the higher risk group, comprising the glandular poorly differentiated subtype (45.8%), including invasive mucinous carcinoma (5.7%), diffuse anaplastic carcinoma (3%), mixed carcinoma (6.7%) (CSS p < 0.0001, DFS p = 0.001). Stage (p < 0.0001), histologic groups (p = 0.001), age >72 years (p = 0.008), and vascular invasion (p = 0.015) were prognostically significant in the multivariate analysis. The combined evaluation of stage/histologic group identified 5-year cancer-specific survival ranging from 87.6% (stage II, lower risk) to 14% (stage IVA, higher risk). Detailed characterization of histologic subtypes contributes to EA/EGJA prognostic prediction.