Multigene Profiling of Circulating Tumor Cells in Esophageal Squamous Cell Carcinoma Identifies Prognostic Cancer Driver Genes Associated with Epithelial-Mesenchymal-Transition Progression and Chemoresistance.

We investigated the clinical significance of CTCs in cancer progression by detecting multiple cancer driver genes associated with epithelial-to-mesenchymal transition (EMT) at the transcript level. The 10-gene panel, comprising CCND1, ECT2, EpCAM, FSCN1, KRT5, KRT18, MET, TFRC, TWIST1, and VEGFC, was established for characterizing CTCs from mouse ESCC xenograft models and clinical ESCC peripheral blood (PB) samples. Correlations between gene expression in CTCs from PB samples (n = 77) and clinicopathological features in ESCC patients (n = 55) were examined. The presence of CTCs at baseline was significantly correlated with tumor size (p = 0.031). The CTC-high patients were significantly correlated with advanced cancer stages (p = 0.013) and distant metastasis (p = 0.029). High mRNA levels of TWIST1 (Hazard Ratio (HR) = 5.44, p = 0.007), VEGFC (HR = 6.67, p < 0.001), TFRC (HR = 2.63, p = 0.034), and EpCAM (HR = 2.53, p = 0.041) at baseline were significantly associated with a shorter overall survival (OS) in ESCC patients. This study also revealed that TWIST1 facilitates EMT and enhances malignant potential by promoting tumor migration, invasion, and cisplatin chemoresistance through the TWIST1-TGFBI-ZEB1 axis in ESCC, highlighting the prognostic and therapeutic potential of TWIST1 in clinical ESCC treatment.

Circulating Tumor DNA Dynamics as Prognostic Markers in Locally Advanced and Metastatic Esophageal Squamous Cell Carcinoma.

Importance: Esophageal squamous cell carcinoma (ESCC) is a deadly disease with frequent recurrence. There are unmet needs for prognostic biomarkers for dynamically monitoring disease progression and detecting minimal residual disease. Objective: To examine whether circulating tumor DNA is clinically useful as a prognostic biomarker for ESCC recurrence and patient survival. Design, Setting, and Participants: This single-center, population-based cohort study consecutively enrolled 147 patients receiving curative (n = 74) or palliative (n = 73) treatment at the surgery and clinical oncology departments of Queen Mary Hospital in Hong Kong from August 1, 2016, to September 31, 2021. Patients were followed up for 2 years. Plasma samples were collected at different longitudinal time points for a prospective circulating tumor DNA (ctDNA) next-generation sequencing profiling study of 77 actionable genes. Intervention: Patients were treated with up-front surgery, neoadjuvant chemoradiotherapy plus surgery with or without adjuvant therapy, or palliative chemotherapy (CT). Main Outcomes and Measures: Detection of circulating tumor DNA (ctDNA), progression-free survival (PFS), and overall survival (OS). Results: A total of 478 serial plasma samples from 147 patients with locoregional or metastatic ESCC were prospectively analyzed. Among the 74 patients in the curative group (median [range] age, 66 [46-85] years; 56 [76.0%] male), 44 (59.5%) relapsed and 36 (48.6%) died. For patients receiving curative surgical treatment, a high ctDNA level (hazard ratio [HR], 7.84; 95% CI, 1.87-32.97; P = .005) and ctDNA alterations (HR, 5.71; 95% CI, 1.81-17.97; P = .003) at 6 months postoperation were independently associated with poor OS. Among patients receiving neoadjuvant chemoradiotherapy, postneoadjuvant ctDNA alterations were associated with poor PFS (HR, 3.16; 95% CI, 1.17-8.52; P = .02). In the 73 patients in the palliative group (median [range] age, 63 [45-82] years; 63 [86.0%] male), 71 (97.3%) had disease relapse and 68 (93.2%) died. Detectable pre-CT NFE2L2 alterations were independently associated with PFS (HR, 2.99; 95% CI, 1.35-6.61; P = .007) and OS (HR, 28.39; 95% CI, 7.26-111.03; P = 1.52 × 10-6), whereas high ctDNA levels (HR, 2.41; 95% CI, 1.18-4.95; P = .02) and alterations in pre-cycle III ctDNA (HR, 1.99; 95% CI, 1.03-3.85; P = .04) showed weaker associations with PFS. Alterations in pre-CT ctDNA were independently associated with OS (HR, 4.46; 95% CI, 1.86-10.69; P = 7.97 × 10-4). Conclusions and Relevance: The findings of this cohort study indicate that prognostic models incorporating ctDNA features are useful in ESCC. Both ctDNA level and NFE2L2 alterations pre-CT and before cycle III were found to be important prognostic factors in palliative groups, and ctDNA alterations after treatment and at 6 months after surgery may define high-risk groups for recurrence in the curative group. High-risk patients can benefit by a timely switch to the next therapeutic options.

A Prospective Study Evaluating the Feasibility and Accuracy of Post-operative Laryngeal Ultrasonography (LUSG) in Assessment of Vocal Cord Function After Esophagectomy.

BACKGROUND: Vocal cord paresis (VCP) is a serious complication after esophagectomy. Conventional diagnosis of VCP relies on flexible laryngoscopy (FL), which is invasive. Laryngeal ultrasonography (LUSG) is non-invasive and convenient. It has provided accurate VC evaluation after thyroidectomy but it is unclear if it is just as accurate following esophagectomy. This prospective study evaluated the feasibility and accuracy of LUSG in VC assessment on day-1 after esophagectomy. METHODS: Consecutive patients from a tertiary teaching hospital who underwent elective esophagectomy were prospectively recruited. All received pre-operative FL, and post-operative LUSG and FL on Day-1, each performed by a blinded, independent assessor. The primary outcomes were feasibility and accuracy of LUSG in the diagnosis of VCP on Day-1 post-esophagectomy. The accuracy of voice assessment (VA) was analyzed. RESULTS: Twenty-six patients were eligible for analysis. The median age was 70 years (66-73). Majority were male (84.6%). Twenty-five (96.2%) received three-phase esophagectomy. Twenty-four (96%) had same-stage anastomosis at the neck. Three (11.5%) developed temporary and one (3.8%) developed permanent unilateral VCP. Overall VC visualization rate by LUSG was 100%; sensitivity, specificity, positive predictive value, negative predictive value (NPV) and accuracy of LUSG were 75.0%, 100%, 100%, 98.0%, 98.1% respectively, and superior to VA. Combining LUSG with VA findings could pick up all VCPs i.e. improved sensitivity and NPV to 100%. CONCLUSION: LUSG is a highly feasible, accurate and non-invasive method to evaluate VC function early after esophagectomy. Post-operative FL may be avoided in patients with both normal LUSG and voice.

Circulating Tumor Cell Enumeration for Serial Monitoring of Treatment Outcomes for Locally Advanced Esophageal Squamous Cell Carcinoma.

We aim to reveal the clinical significance and potential usefulness of dynamic monitoring of CTCs to track therapeutic responses and improve survival for advanced ESCC patients. Peripheral blood (PB) (n = 389) and azygos vein blood (AVB) (n = 13) samplings were recruited prospectively from 88 ESCC patients undergoing curative surgery from 2017 to 2022. Longitudinal CTC enumeration was performed with epithelial (EpCAM/pan-cytokeratins/MUC1) and mesenchymal (vimentin) markers at 12 serial timepoints at any of the pre-treatment, all of the post-treatments/pre-surgery, post-surgery follow-ups for 3-year, and relapse. Longitudinal real-time CTC analysis in PB and AVB suggests more CTCs are released early at pre-surgery and 3-month post-surgery into the circulation from the CTRT group compared to the up-front surgery group. High CTC levels at pre-treatments, 1-/3-month post-surgery, unfavorable changes of CTC levels between all post-treatment/pre-surgery and 1-month or 3-month post-surgery (Hazard Ratio (HR) = 6.662, p < 0.001), were independent prognosticators for curative treatment. The unfavorable pre-surgery CTC status was independent prognostic and predictive for neoadjuvant treatment efficacy (HR = 3.652, p = 0.035). The aggressive CTC clusters were more frequently observed in AVB compared to PB. Its role as an independent prognosticator with relapse was first reported in ESCC (HR = 2.539, p = 0.068). CTC clusters and longitudinal CTC monitoring provide useful prognostic information and potential predictive biomarkers to help guide clinicians in improving disease management.

Clonal relationship and alcohol consumption-associated mutational signature in synchronous hypopharyngeal tumours and oesophageal squamous cell carcinoma.

BACKGROUND: The patients with dual oesophageal squamous cell carcinoma (ESCC) and hypopharyngeal cancer (HPC) have poor prognosis; their underlying genetic pathogenesis is unclear. We hypothesise that development of synchronous ESCC/HPC depends on multicentricity or independent origin, rather than multifocality due to local or lateral spreading. METHOD: Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumour heterogeneity (ITH) in 62 tumour regions from eight dual ESCC/HPC and ten ESCC patients. RESULTS: All synchronous ESCC/HPC patients had COSMIC 16 mutation signatures, compared to only 40% ESCC in the current study (p = 0.013) and public data set (n = 165, p = 0.003). This alcohol consumption-related mutation signature 16, commonly involved in multiple alcohol-related cancers, was significantly associated with drinking and alcohol metabolism-related ADH1B rs1229984. The mutational landscape and copy number profiles were completely distinct between the two primary tumours; clonality analysis further suggested the two primary tumours shared no or only one clone accompanying independent subclone evolution. M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumours, compared to traditional sequencing analysis based on single biopsy strategy. Patients with high ITH assessed by cancer cell fraction analysis after M-WES were significantly associated with both relapse and survival. CONCLUSIONS: Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. Collectively, our findings support multicentric independent clonal evolution, the field cancerisation theory, and suggest novel insights implicating an aetiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.

Synergistic Interaction of Polycyclic Aromatic Hydrocarbons, Phthalate Esters, or Phenol on DNA Adduct Formation by Aristolochic Acid I: Insights into the Etiology of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with chronic exposure to aristolochic acids (AAs) through AA-contaminated food being one of the major etiological mechanisms. However, the bulk of previous research has only focused on investigating the possible roles of individual pollutants in disease development and the etiological mechanism of BEN remains controversial. In this study, we investigated the exposure concentration and duration dependence of coexposure to phthalate esters and lignite coal-derived phenol and polycyclic aromatic hydrocarbons (PAHs) on the metabolism and DNA adduct formation of aristolochic acid I (AAI). Results showed that both the metabolic activation and DNA adduct formation of AAI in cultured human kidney cells were affected by their coexposure to the above-mentioned environmental pollutants. Furthermore, our results suggest that chemicals leached from lignite coal likely played a role by triggering AA-activating enzymes to produce more of the promutagenic DNA adducts, thus further elevating the nephrotoxicity and carcinogenicity of AAs and increasing the risk of BEN. It is believed that the results of this study provide a better understanding of the etiological mechanism of BEN and offer insights into methods and policies to lower the risk of this devastating disease.

Quantitation of Protein Adducts of Aristolochic Acid I by Liquid Chromatography-Tandem Mass Spectrometry: A Novel Method for Biomonitoring Aristolochic Acid Exposure.

Emerging evidence suggests that chronic exposure to aristolochic acids (AAs) is one of the etiological pathways leading to chronic kidney disease (CKD). Due to the traditional practice of herbal medicine and AA-containing plants being used extensively as medicinal herbs, over 100 million East Asians are estimated to be at risk of AA poisoning. Given that the chronic nephrotoxicity of AAs only manifests itself after decades of exposure, early diagnosis of AA exposure could allow for timely intervention and disease risk reduction. However, an early detection method is not yet available, and diagnosis can only be established at the end stage of CKD. The goal of this study was to develop a highly sensitive and selective method to quantitate protein adducts of aristolochic acid I (AAI) as a biomarker of AA exposure. The method entails the release of protein-bound aristolactam I (ALI) by heat-assisted alkaline hydrolysis, extraction of ALI, addition of internal standard, and quantitation by liquid chromatography-tandem mass spectrometric analysis. Accuracy and precision of the method were critically evaluated using a synthetic ALI-containing glutathione adduct. The validated method was subsequently used to detect dose-dependent formation of ALI-protein adducts in human serum albumin exposed to AAI and in proteins isolated from the tissues and sera of AAI-exposed rats. Our time-dependent study showed that ALI-protein adducts remained detectable in rats even at 28 days postdosing. It is anticipated that the developed method will fill the technical gap in diagnosing AA intoxication and facilitate the biomonitoring of human exposures to AAs.

Comparison of laparoscopic versus open gastrectomy for gastric cancer.

BACKGROUND: This study aimed to compare the short-term and long-term outcomes of laparoscopic gastrectomy (LG) and open gastrectomy (OG) for gastric cancer in a tertiary referral center in Hong Kong. METHODS: Two hundred and ninety-four consecutive patients with gastric cancer who underwent radical gastrectomy with curative intent between January 2008 and December 2015 were analyzed. Data was prospectively collected and reviewed. Propensity score matching was applied at a ratio of 1:1 to compare the OG and LG groups. RESULTS: After propensity score matching, operation duration (294.7 vs 231.8min, P < 0.01) was significantly longer while estimated blood loss (191.6 vs 351.0 ml, P = 0.01) was significantly less in LG group compared with OG. There were no significant differences in postoperative complications and mortality between LG and OG groups (postoperative complication rate, 35.2% vs 40.7%, P = 0.69; 90-day mortality rate, 1.9% vs 3.7%, P = 1.00). Three-year OS and 3-yr DFS of patients who underwent LG was not inferior to that of patients who had OG (P = 0.34; P = 0.51). However, there were significantly more peritoneal recurrences among the OG group than LG group (P < 0.01). CONCLUSIONS: LG has comparable outcomes for gastric cancer, even in advanced tumors. We could appropriately increase the proportion of laparoscopic gastrectomy for gastric cancer.

Real-world Scenario: CROSS Regimen as Preoperative Therapy for Oesophageal Squamous Cell Carcinoma.

BACKGROUND: Preoperative chemoradiation (CROSS regimen) has been widely adopted worldwide. The survival advantage imparted is especially impressive for oesophageal squamous cell carcinoma (OSCC). This study aimed at investigating the efficacy of the CROSS regimen in real-world scenario. METHODS: This is a retrospective study of all patients with OSCC intended for preoperative treatment using the CROSS regimen during 2012-2017. Patients were divided into two groups: those within the selection criteria in the CROSS trial and those beyond criteria, namely age > 75 years old, tumour length > 8 cm or clinical M1 stage of lymph node involvement (AJCC 6th edition). Clinical outcome and survival data were compared. RESULTS: Eighty-eight patients were included. There were 46 patients in the "CROSS eligible" group and 42 in the "CROSS ineligible" group. By intention-to-treat, the estimated median survival was 24.2 months vs. 12.7 months, respectively (p = 0.047). The results were much inferior compared to that published in the original CROSS trial. Univariable and multivariable analyses showed tumour length and resectability as independent variables affecting survival. DISCUSSION: In a real-world scenario, the clinical outcome remains suboptimal and the excellent results in the trial setting were not reproducible in this Asian cohort. Patient selection is one key element accountable for the difference. The efficacy of the CROSS regimen may not be adequate for patients with more advanced disease. The optimal multimodal therapy for this group of patients remains undefined.

Impact of Extracapsular Extension of Lymph Node in Adenocarcinoma of the Stomach.

BACKGROUND: Extracapsular extension (ECE) of lymph node may have important prognostic impact for patients with adenocarcinoma of the stomach, but it generally is ignored in staging systems and prognostic models. This study aimed to examine the impact that ECE of lymph node has on prognosis for patients with adenocarcinoma of the stomach. METHODS: The study analyzed 321 consecutive patients with gastric cancer who underwent radical gastrectomy between January 2008 and December 2015. None of these patients had distant metastases. Lymph node metastases were found in 187 patients. The ECE grade was evaluated according to the previously described system used in head and neck cancers. Deposits of cancer cells in sub-serosal fat without a recognizable lymph node were classified as ECE grade 4. Survival outcomes were compared using Kaplan-Meier and Cox regression analyses. A nomogram was constructed using identified significant prognostic factors. The predictive accuracy and model performance were measured by the concordance index (C-index). RESULTS: Patients with ECE(+) showed significantly worse 3-year overall survival (OS) and disease-free survival (DFS) than those without ECE. In the sensitivity analysis, ECE had independent prognostic value for both 3-year OS and 3-year DFS, whereas ECE grading showed little impact on mortality trend or disease progression trend. The ECE-based nomogram showed a significantly higher C-index than the pathological tumor and node staging (pTN) staging system. CONCLUSIONS: The adverse prognostic impact of ECE was validated. Sub-serosal tumor deposits without recognizable lymph node tissue are recommended for inclusion in the ECE definition. A nomogram involving ECE could provide better individual prediction of survival for patients with lymph node-positive gastric cancer.

Occurrence and Environmental Stability of Aristolochic Acids in Groundwater Collected from Serbia: Links to Human Exposure and Balkan Endemic Nephropathy.

Aristolochic acids (AAs) have been known as potent nephrotoxins since the use of AA-containing herbal medicines was linked with a series of sporadic renal fibrotic nephropathy cases, and yet an estimated 100 million people worldwide are still at risk today because of continued use of similar medicines. However, a similar nephropathic condition is endemic in the rural Balkan regions (e.g., Serbian farming villages) and AAs were again found to be the causative agents. In the case of this Balkan endemic nephropathy, AAs were found to have originated from a widespread local weed Aristolochia clematitis L. In this study, we tested the hypothesis that AAs released from decomposition of A. clematitis were also being leached into groundwater, thus polluting the drinking water of local residents. We initiated the study by developing a dispersive solid-phase extraction-based sample preparation method for water samples suspected of AA contamination. The validated method was then coupled with a liquid chromatography-tandem mass spectrometric method to measure AAs in groundwater samples collected from Serbia. Our study revealed for the first time that groundwater in Serbia is extensively contaminated with AA-I, at ng/L levels. Results also showed that AAs are long-lived water contaminants, with no observable concentration changes over a 2-month period of sample storage.