The pluripotency factor NANOG contributes to mesenchymal plasticity and is predictive for outcome in esophageal adenocarcinoma.

BACKGROUND: Despite the advent of neoadjuvant chemoradiotherapy (CRT), overall survival rates of esophageal adenocarcinoma (EAC) remain low. A readily induced mesenchymal transition of EAC cells contributes to resistance to CRT. METHODS: In this study, we aimed to chart the heterogeneity in cell state transition after CRT and to identify its underpinnings. A panel of 12 esophageal cultures were treated with CRT and ranked by their relative epithelial-mesenchymal plasticity. RNA-sequencing was performed on 100 pre-treatment biopsies. After RNA-sequencing, Ridge regression analysis was applied to correlate gene expression to ranked plasticity, and models were developed to predict mesenchymal transitions in patients. Plasticity score predictions of the three highest significant predictive models were projected on the pre-treatment biopsies and related to clinical outcome data. Motif enrichment analysis of the genes associated with all three models was performed. RESULTS: This study reveals NANOG as the key associated transcription factor predicting mesenchymal plasticity in EAC. Expression of NANOG in pre-treatment biopsies is highly associated with poor response to neoadjuvant chemoradiation, the occurrence of recurrences, and median overall survival difference in EAC patients (>48 months). Perturbation of NANOG reduces plasticity and resensitizes cell lines, organoid cultures, and patient-derived in vivo grafts. CONCLUSIONS: In conclusion, NANOG is a key transcription factor in mesenchymal plasticity in EAC and a promising predictive marker for outcome.

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Although chemotherapy combined with radiotherapy (chemoradiotherapy) followed by surgery has improved survival, tumor recurrence and metastatic disease (that has spread to other parts of the body) are often observed after several months. In this study, we assessed the effect of chemoradiotherapy on esophageal cells in the lab to predict the effect in patients with esophageal cancer. To investigate this, genes were assessed from 12 different cell lines and 100 patient tissues. We revealed that levels of one of the genes, NANOG, associates with poor response in patients. NANOG could be a promising marker to predict outcome in patients with esophageal cancer. This knowledge might help clinicians to treat patients with esophageal cancer appropriately, or may lead to new or optimized treatments.

Exploiting a subtype-specific mitochondrial vulnerability for successful treatment of colorectal peritoneal metastases.

Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.

Multi-modal cell-free DNA genomic and fragmentomic patterns enhance cancer survival and recurrence analysis.

The structure of cell-free DNA (cfDNA) is altered in the blood of patients with cancer. From whole-genome sequencing, we retrieve the cfDNA fragment-end composition using a new software (FrEIA [fragment end integrated analysis]), as well as the cfDNA size and tumor fraction in three independent cohorts (n = 925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% cancer samples using at least one cfDNA measure, including 64% early-stage cancer (n = 220). cfDNA detection correlates with a shorter overall (p = 0.0086) and recurrence-free (p = 0.017) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n = 396 cancer, 90 controls) achieve a detection accuracy of 82% and area under the receiver operating characteristic curve of 0.96. In conclusion, harnessing the biological features of cfDNA can improve, at no extra cost, the diagnostic performance of liquid biopsies.

Real-time analysis of the cancer genome and fragmentome from plasma and urine cell-free DNA using nanopore sequencing.

Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.

The landscape of cell-free mitochondrial DNA in liquid biopsy for cancer detection.

BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.

Local irradiation of patient-derived tumors in immunodeficient mice.

Severe combined immunodeficient mice are typically used for xenografting experiments and show reliable tumor engraftment; however, their Prkdscid mutation renders them highly sensitive to irradiation. Here, we describe a protocol that allows safe local irradiation of tumor xenografts in immunodeficient mice. We detail the steps for the establishment and handling of patient-derived cancer cultures, subcutaneous injection of cancer cells on the mouse hind limb, localized irradiation in mice, tumor monitoring, and tumor characterization via histological and immunohistochemical assessment. For complete details on the use and execution of this protocol, please refer to Dings et al. (2022).1.

The molecular biology of peritoneal metastatic disease.

Peritoneal metastases are a common form of tumor cell dissemination in gastrointestinal malignancies. Peritoneal metastatic disease (PMD) is associated with severe morbidity and resistance to currently employed therapies. Given the distinct route of dissemination compared with distant organ metastases, and the unique microenvironment of the peritoneal cavity, specific tumor cell characteristics are needed for the development of PMD. In this review, we provide an overview of the known histopathological, genomic, and transcriptomic features of PMD. We find that cancers representing the mesenchymal subtype are strongly associated with PMD in various malignancies. Furthermore, we discuss the peritoneal niche in which the metastatic cancer cells reside, including the critical role of the peritoneal immune system. Altogether, we show that PMD should be regarded as a distinct disease entity, that requires tailored treatment strategies.

Perturbation of Copper Homeostasis Sensitizes Cancer Cells to Elevated Temperature.

Temporary elevation of tumor temperature, also known as hyperthermia, is a safe and well-tolerated treatment modality. The efficacy of hyperthermia can be improved by efficient thermosensitizers, and various candidate drugs, including inhibitors of the heat stress response, have been explored in vitro and in animal models, but clinically relevant thermosensitizers are lacking. Here, we employ unbiased in silico approaches to uncover new mechanisms and compounds that could be leveraged to increase the thermosensitivity of cancer cells. We then focus on elesclomol, a well-performing compound, which amplifies cell killing by hyperthermia by 5- to 20-fold in cell lines and outperforms clinically applied chemotherapy when combined with hyperthermia in vitro. Surprisingly, our findings suggest that the thermosensitizing effects of elesclomol are independent of its previously reported modes of action but depend on copper shuttling. Importantly, we show that, like elesclomol, multiple other copper shuttlers can thermosensitize, suggesting that disturbing copper homeostasis could be a general strategy for improving the efficacy of hyperthermia.

Estrogen-related receptor alpha drives mitochondrial biogenesis and resistance to neoadjuvant chemoradiation in esophageal cancer.

Neoadjuvant chemoradiotherapy (nCRT) improves outcomes in resectable esophageal adenocarcinoma (EAC), but acquired resistance precludes long-term efficacy. Here, we delineate these resistance mechanisms. RNA sequencing on matched patient samples obtained pre-and post-neoadjuvant treatment reveal that oxidative phosphorylation was the most upregulated of all biological programs following nCRT. Analysis of patient-derived models confirms that mitochondrial content and oxygen consumption strongly increase in response to nCRT and that ionizing radiation is the causative agent. Bioinformatics identifies estrogen-related receptor alpha (ESRRA) as the transcription factor responsible for reprogramming, and overexpression and silencing of ESRRA functionally confirm that its downstream metabolic rewiring contributes to resistance. Pharmacological inhibition of ESRRA successfully sensitizes EAC organoids and patient-derived xenografts to radiation. In conclusion, we report a profound metabolic rewiring following chemoradiation and demonstrate that its inhibition resensitizes EAC cells to radiation. These findings hold broader relevance for other cancer types treated with radiation as well.

Molecular characterization of colorectal cancer related peritoneal metastatic disease.

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.

Serum-based measurements of stromal activation through ADAM12 associate with poor prognosis in colorectal cancer.

BACKGROUND: Recently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal activation, A Disintegrin and Metalloprotease 12 (ADAM12) in colorectal cancer (CRC). METHODS: Using gene expression databases we investigated ADAM12 expression in CRC and delineated the source of ADAM12 expression. The clinical value of ADAM12 was retrospectively assessed in the CAIRO2 trial in metastatic CRC with 235 patients (31% of total cohort), and an independent rectal cancer cohort (n = 20). RESULTS: ADAM12 is expressed by activated CRC associated fibroblasts. In the CAIRO2 trial cohort, ADAM12 serum levels were prognostic (ADAM12 low versus ADAM12 high; median OS 25.3 vs. 17.1 months, HR 1.48 [95% CI 1.11-1.96], P = 0.007). The prognostic potential was specifically high for metastatic rectal cancer (HR 1.78 [95% CI 1.06-3.00], P = 0.030) and mesenchymal subtype tumors (HR 2.12 [95% CI 1.25-3.60], P = 0.004). ADAM12 also showed potential for predicting recurrence in an exploratory analysis of non-metastatic rectal cancers. CONCLUSIONS: Here we describe a non-invasive marker for activated stroma in CRC which associates with poor outcome, especially for primary cancers located in the rectum.

Tuberculosis Treatment Outcome and Drug Resistance in Lambaréné, Gabon: A Prospective Cohort Study.

Despite overall global progress in tuberculosis (TB) control, TB remains one of the deadliest communicable diseases. This study prospectively assessed TB epidemiology in Lambaréné, Gabon, a Central African country ranking 10th in terms of TB incidence rate in the 2014 World Health Organization TB report. In Lambaréné, between 2012 and 2014, 201 adult and pediatric TB patients were enrolled and followed up; 66% had bacteriologically confirmed TB and 95% had pulmonary TB. The human immunodeficiency virus (HIV) coinfection rate was 42% in adults and 16% in children. Mycobacterium tuberculosis and Mycobacterium africanum were identified in 82% and 16% of 108 culture-confirmed TB cases, respectively. Isoniazid (INH) and streptomycin yielded the highest resistance rates (13% and 12%, respectively). The multidrug resistant TB (MDR-TB) rate was 4/91 (4%) and 4/13 (31%) in new and retreatment TB cases, respectively. Treatment success was achieved in 53% of patients. In TB/HIV coinfected patients, mortality rate was 25%. In this setting, TB epidemiology is characterized by a high rate of TB/HIV coinfection and low treatment success rates. MDR-TB is a major public health concern; the need to step-up in-country diagnostic capacity for culture and drug susceptibility testing as well as access to second-line TB drugs urgently requires action.