Master corepressor inactivation through multivalent SLiM-induced polymerization mediated by the oncogene suppressor RAI2.

While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.

PIK3CA Hotspot Mutations in Saliva as a Diagnostic Marker in Oral Squamous Cell Carcinoma Patients.

BACKGROUND/AIM: This study aimed at the analogous detection of PIK3CA mutations, common in oral squamous cell carcinoma (OSCC), in matched tumor and saliva samples. PATIENTS AND METHODS: Tissue and saliva samples were obtained from 29 patients diagnosed with primary OSCC. Saliva samples were obtained preoperatively; tissue specimens were acquired during tumor resection. Tumor DNA was extracted from both tissue and saliva samples. All samples were controlled for DNA quantity and quality and genetic matching of sample pairs was confirmed using the iPlex Pro Exome QC Panel. Variant detection was performed using the MassARRAY® System, a mass-spectrometry based detection system. Mutational analysis in tissue tumor DNA was made using the multiplexed ClearSEEK™ PIK3CA v1.0 Panel covering 20 hotspot mutations in PIK3CA. In saliva samples, variants were analyzed using both the ClearSEEK™ and the UltraSEEK® Lung v1.1 Panel, with a higher limit of detection but covering less PIK3CA variants. RESULTS: Overall, a PIK3CA variant was found in seven of the 29 tumor tissue samples (24%) by ClearSEEK™; UltraSEEK® additionally confirmed the variant in four of these seven positive samples. Of the three variants not detected by UltraSEEK®, two were not included in the panel and one was included but not detected. Of the seven variants found in tissue, five could also be detected in the matching saliva samples (71%), either by utilizing ClearSEEK™ or UltraSEEK® Conclusion: The detection of PIK3CA hotspot mutations in OSCC and their simultaneous occurrence in saliva underline the potential benefit of liquid biopsies for non-invasive cancer detection and follow-up care of OSCC patients.

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis.

BACKGROUND: Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the "metastasis-suppressor" effect of HERC5, with a focus on mitochondrial metabolism pathways. METHODS: We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. RESULTS: Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. CONCLUSIONS: Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

Frequency and Prognostic Value of Circulating Tumor Cells in Cancer of Unknown Primary.

BACKGROUND: Cancer of unknown primary (CUP) is defined as a primary metastatic malignancy, in which the primary tumor remains elusive in spite of a comprehensive diagnostic workup. The frequency and prognostic value of circulating tumor cells (CTCs), which are considered to be the source of metastasis, has not yet been systematically evaluated in CUP. METHODS: A total of 110 patients with a confirmed diagnosis of CUP according to the European Society for Medical Oncology (ESMO) guidelines, who presented to our clinic between July 2021 and May 2023, provided blood samples for CTC quantification using CellSearch methodology. CTC counts were correlated with demographic, clinical, and molecular data generated by comprehensive genomic profiling of tumor tissue. RESULTS: CTCs were detected in 26% of all patients at initial presentation to our department. The highest CTC frequency was observed among patients with unfavorable CUP (35.5%), while patients with single-site/oligometastatic CUP harbored the lowest CTC frequency (11.4%). No statistically significant association between CTC positivity and the number of affected organs (P = 0.478) or disease burden (P = 0.120) was found. High CTC levels (≥5 CTCs/7.5 mL; 12/95 analyzed patients) predicted for adverse overall survival compared to negative or low CTC counts (6-months overall survival rate 90% vs 32%, log-rank P < 0.001; HR 5.43; 95% CI 2.23-13.2). CTC dynamics were also prognostic for overall survival by landmark analysis (log-rank P < 0.001, HR 10.2, 95% CI 1.95-52.9). CONCLUSIONS: CTC frequency is a strong, independent predictor of survival in patients with CUP. CTC quantification provides a useful prognostic tool in the management of these patients.

Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia.

BACKGROUND: More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. METHODS: In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. RESULTS: The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. CONCLUSIONS: This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.

RNF40 epigenetically modulates glycolysis to support the aggressiveness of basal-like breast cancer.

Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat due to the lack of targeted therapies. Cancer stem cells (CSCs) are strongly enriched in TNBC lesions and are responsible for the rapid development of chemotherapy resistance and metastasis. Ubiquitin-based epigenetic circuits are heavily exploited by CSCs to regulate gene transcription and ultimately sustain their aggressive behavior. Therefore, therapeutic targeting of these ubiquitin-driven dependencies may reprogram the transcription of CSC and render them more sensitive to standard therapies. In this work, we identified the Ring Finger Protein 40 (RNF40) monoubiquitinating histone 2B at lysine 120 (H2Bub1) as an indispensable E3 ligase for sustaining the stem-cell-like features of the growing mammary gland. In addition, we found that the RNF40/H2Bub1-axis promotes the CSC properties and drug-tolerant state by supporting the glycolytic program and promoting pro-tumorigenic YAP1-signaling in TNBC. Collectively, this study unveils a novel tumor-supportive role of RNF40 and underpins its high therapeutic value to combat the malignant behavior of TNBC.

The role of the desmosomal protein desmocollin 2 in tumour progression in triple negative breast cancer patients.

BACKGROUND: The disruption of epithelial features represents a critical step during breast cancer spread. In this context, the dysregulation of desmosomal proteins has been associated with malignant progression and metastasis formation. Curiously, both tumour suppressive and pro-metastatic roles have been attributed to desmosomal structures in different cancer entities. In the present study, we describe the pro-metastatic role of the desmosomal protein desmocollin 2 (DSC2) in breast cancer. METHODS: We analysed the prognostic role of DSC2 at mRNA and protein level using microarray data, western blot analysis and immunohistochemistry. Functional consequences of DSC2 overexpression and DSC2 knock down were investigated in the triple negative breast cancer (TNBC) cell line MDA-MB-231 and its brain-seeking subline MDA-MB-231-BR, respectively in vitro and in vivo. RESULTS: We found a significantly higher DSC2 expression in the more aggressive molecular subtypes HER2-positive and TNBC than in luminal breast cancers, as well as a significant correlation between increased DSC2 expression and a shorter disease-free-also in multivariate analysis-and overall survival. Additionally, a significant association between DSC2 expression in the primary tumour and an increased frequency of cerebral and lung metastasis could be observed. In vitro, ectopic DSC2 expression or DSC2 down-regulation in MDA-MB-231 and MDA-MB-231-BR led to a significant tumour cell aggregation increase and decrease, respectively. Furthermore, tumour cells displaying higher DSC2 levels showed increased chemoresistance in 3D structures, but not 2D monolayer structures, suggesting the importance of cell aggregation as a means for reduced drug diffusion. In an in vivo brain dissemination xenograft mouse model, reduced expression of DSC2 in the brain-seeking TNBC cells led to a decreased amount of circulating tumour cells/clusters and, in turn, to fewer and smaller brain metastatic lesions. CONCLUSION: We conclude that high DSC2 expression in primary TNBC is associated with a poorer prognosis, firstly by increasing tumour cell aggregation, secondly by reducing the diffusion and effectiveness of chemotherapeutic agents, and, lastly, by promoting the circulation and survival of tumour cell clusters, each of which facilitates distant organ colonisation.

Comparative evaluation of PD-L1 expression in cytology imprints, circulating tumour cells and tumour tissue in non-small cell lung cancer patients.

Alternative sources of tumour information need to be explored in patients with non-small cell lung cancer (NSCLC). Here, we compared programmed cell death ligand 1 (PD-L1) expression on cytology imprints and circulating tumour cells (CTCs) with PD-L1 tumour proportion score (TPS) from immunohistochemistry staining of tumour tissue from patients with NSCLC. We evaluated PD-L1 expression using a PD-L1 antibody (28-8) in representative cytology imprints, and tissue samples from the same tumour. We report good agreement rates on PD-L1 positivity (TPS ≥ 1%) and high PD-L1 expression (TPS ≥ 50%). Considering high PD-L1 expression, cytology imprints showed a PPV of 64% and a NPV of 85%. CTCs were detected in 40% of the patients and 80% of them were PD-L1+ . Seven patients with PD-L1 expression of < 1% in tissue samples or cytology imprints had PD-L1+ CTCs. The addition of PD-L1 expression in CTCs to cytology imprints markedly improved the prediction capacity for PD-L1 positivity. A combined analysis of cytological imprints and CTCs provides information on the tumoural PD-L1 status in NSCLC patients, which might be used when no tumor tissue is available.

Peripheral and Portal Venous KRAS ctDNA Detection as Independent Prognostic Markers of Early Tumor Recurrence in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: KRAS circulating tumor DNA (ctDNA) has shown biomarker potential for pancreatic ductal adenocarcinoma (PDAC) but has not been applied in clinical routine yet. We aim to improve clinical applicability of ctDNA detection in PDAC and to study the impact of blood-draw site and time point on the detectability and prognostic role of KRAS mutations. METHODS: 221 blood samples from 108 PDAC patients (65 curative, 43 palliative) were analyzed. Baseline peripheral and tumor-draining portal venous (PV), postoperative, and follow-up blood were analyzed and correlated with prognosis. RESULTS: Significantly higher KRAS mutant detection rates and copy numbers were observed in palliative compared to curative patients baseline blood (58.1% vs 24.6%; P = 0.002; and P < 0.001). Significantly higher KRAS mutant copies were found in PV blood compared to baseline (P < 0.05) samples. KRAS detection in pre- and postoperative and PV blood were significantly associated with shorter recurrence-free survival (all P < 0.015) and identified as independent prognostic markers. KRAS ctDNA status was also an independent unfavorable prognostic factor for shorter overall survival in both palliative and curative cohorts (hazard ratio [HR] 4.9, P = 0.011; HR 6.9, P = 0.008). CONCLUSIONS: KRAS ctDNA detection is an independent adverse prognostic marker in curative and palliative PDAC patients-at all sites of blood draw and a strong follow-up marker. The most substantial prognostic impact was seen for PV blood, which could be an effective novel tool for identifying prognostic borderline patients-guiding future decision-making on neoadjuvant treatment despite anatomical resectability. In addition, higher PV mutant copy numbers contribute to an improved technical feasibility.

Clinical determinants impacting overall survival of patients with operable brain metastases from non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is currently the leading cause of cancer-related death worldwide, and the incidence of brain metastases (BM) in NSCLC patients is continuously increasing. The recent improvements of systemic treatment in NSCLC necessitate continuous updates on prognostic subgroups and factors determining overall survival (OS). In order to improve clinical decision-making in tumor boards, we investigated the clinical determinants affecting survival in patients with resectable NSCLC BM. A retrospective analysis was conducted of NSCLC patients with surgically resectable BM treated in our institution between 01/2015 and 12/2020. The relevant clinical factors affecting survival identified by univariate analysis were included in a multivariate logistic regression model. Overall, 264 patients were identified, with a mean age of 62.39 ± 9.98 years at the initial diagnosis of NSCLC BM and OS of 23.22 ± 1.71 months. The factors that significantly affected OS from the time of primary tumor diagnosis included the systemic metastatic load (median: 28.40 ± 4.82 vs. 40.93 ± 11.18 months, p = 0.021) as well as a number of BM <2 (median: 17.20 ± 2.52 vs. 32.53 ± 3.35 months, p = 0.014). When adjusted for survival time after neurosurgical intervention, a significant survival benefit was found in patients <60 years (median 16.13 ± 3.85 vs. 9.20 ± 1.39 months, p = 0.011) and, among others, patients without any concurrent systemic metastases at time of NSCLC BM diagnosis. Our data shows that the number of BM (singular/solitary), the Karnofsky Performance Status, gender, and age but not localization (infra-/supratentorial), mass-edema index or time to BM occurrence impact OS, and postsurgical survival in NSCLC BM patients. Additionally, our study shows that patients in prognostically favorable clinical subgroups an OS, which differs significantly from current statements in literature. The described clinically relevant factors may improve the understanding of the risks and the course of this disease and Faid future clinical decision making in tumor boards.

Circulating tumor cells and extracellular vesicles as liquid biopsy markers in neuro-oncology: prospects and limitations.

For many tumor entities, tumor biology and response to therapy are reflected by components that can be detected and captured in the blood stream. The so called "liquid biopsy" has been stratified over time into the analysis of circulating tumor cells (CTC), extracellular vesicles (EVs), and free circulating components such as cell-free nucleic acids or proteins. In neuro-oncology, two distinct areas need to be distinguished, intrinsic brain tumors and tumors metastatic to the brain. For intrinsic brain tumors, specifically glioblastoma, CTCs although present in low abundance, contain highly relevant, yet likely incomplete biological information for the whole tumor. For brain metastases, CTCs can have clinical relevance for patients especially with oligometastatic disease and brain metastasis in cancers like breast and lung cancer. EVs shed from the tumor cells and the tumor environment provide complementary information. Sensitive technologies have become available that are able to detect both, CTCs and EVs in the peripheral blood of patients with intrinsic and metastatic brain tumors despite the blood brain barrier. In reference to glioblastoma EVs, being shed by tumor cells and microenvironment and being more diffusible than CTCs may yield a more complete reflection of the whole tumor compared to low-abundance CTCs representing only a fraction of the multiclonal tumor heterogeneity. We here review the emerging aspects of CTCs and EVs as liquid biopsy biomarkers in neuro-oncology.

Circulating Cancer Associated Macrophage-like Cells as a Potential New Prognostic Marker in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Circulating Cancer Associated Macrophage-like cells (CAMLs) have been described as novel liquid biopsy analytes and unfavorable prognostic markers in some tumor entities, with scarce data for Pancreatic Ductal Adenocarcinomas (PDAC). METHODS: Baseline and follow-up blood was drawn from resected curative (n = 36) and palliative (n = 19) PDAC patients. A microfluidic size-based cell enrichment approach (ParsortixTM) was used for CAML detection, followed by immunofluorescence staining using pan-keratin, CD14, and CD45 antibodies to differentiate between CAMLs, circulating tumor cells (CTCs), and leukocytes. RESULTS: CAMLs were detectable at baseline in 36.1% of resected patients and 47.4% of palliative PDAC patients. CAML detection was tumor stage independent. Follow-up data indicated that detection of CAMLs (in 45.5% of curative patients) was an independent prognostic factor for shorter recurrence-free survival (RFS) (HR: 4.3, p = 0.023). Furthermore, a combined analysis with CTCs showed the detectability of at least one of these cell populations in 68.2% of resected patients at follow-up. The combined detection of CAMLs and CTCs was also significantly associated with short RFS (HR: 8.7, p = 0.003). CONCLUSIONS: This pilot study shows that detection of CAMLs in PDAC patients can provide prognostic information, either alone or even more pronounced in combination with CTCs, which indicates the power of liquid biopsy marker analyses.

Characterization of RARRES1 Expression on Circulating Tumor Cells as Unfavorable Prognostic Marker in Resected Pancreatic Ductal Adenocarcinoma Patients.

BACKGROUND: In pancreatic ductal adenocarcinoma (PDAC), the characterization of circulating tumor cells (CTCs) opens new insights into cancer metastasis as the leading cause of cancer-related death. Here, we focused on the expression of retinoic acid receptor responder 1 (RARRES1) on CTCs as a novel marker for treatment failure and early relapse. METHODS: The stable isotope labeling of amino acids in cell culture (SILAC)-approach was applied for identifying and quantifying new biomarker proteins in PDAC cell lines HPDE and its chemoresistant counterpart, L3.6pl-Res. Fifty-five baseline and 36 follow-up (FUP) peripheral blood samples were processed via a marker-independent microfluidic-based CTC detection approach using RARRES1 as an additional marker. RESULTS: SILAC-based proteomics identified RARRES1 as an abundantly expressed protein in more aggressive chemoresistant PDAC cells. At baseline, CTCs were detected in 25.5% of all PDAC patients, while FUP analysis (median: 11 months FUP) showed CTC detection in 45.5% of the resected patients. CTC positivity (≥3 CTC) at FUP was significantly associated with short recurrence-free survival (p = 0.002). Furthermore, detection of RARRES1 positive CTCs was indicative of an even earlier relapse after surgery (p = 0.001). CONCLUSIONS: CTC detection in resected PDAC patients during FUP is associated with a worse prognosis, and RARRES1 expression might identify an aggressive subtype of CTCs that deserves further investigation.

Circulating tumor cell-blood cell crosstalk: Biology and clinical relevance.

Circulating tumor cells (CTCs) are the seeds of distant metastasis, and the number of CTCs detected in the blood of cancer patients is associated with a worse prognosis. CTCs face critical challenges for their survival in circulation, such as anoikis, shearing forces, and immune surveillance. Thus, understanding the mechanisms and interactions of CTCs within the blood microenvironment is crucial for better understanding of metastatic progression and the development of novel treatment strategies. CTCs interact with different hematopoietic cells, such as platelets, red blood cells, neutrophils, macrophages, natural killer (NK) cells, lymphocytes, endothelial cells, and cancer-associated fibroblasts, which can affect CTC survival in blood. This interaction may take place either via direct cell-cell contact or through secreted molecules. Here, we review interactions of CTCs with blood cells and discuss the potential clinical relevance of these interactions as biomarkers or as targets for anti-metastatic therapies.

Combined Liquid Biopsy Methylation Analysis of CADM1 and MAL in Cervical Cancer Patients.

Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.

Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer.

BACKGROUND: Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer. METHODS: We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes. RESULTS: Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1. CONCLUSIONS: The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells.

Tumor-Stroma Interaction in PDAC as a New Approach for Liquid Biopsy and its Potential Clinical Implications.

The extremely poor prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) has remained unchanged for decades. As a hallmark of PDAC histology, the distinct desmoplastic response in the tumor microenvironment is considered a key factor exerting pro- and antitumor effects. Increasing emphasis has been placed on cancer-associated fibroblasts (CAFs), whose heterogeneity and functional diversity is reflected in the numerous subtypes. The myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs) and antigen presenting CAFs (apCAFs) are functionally divergent CAF subtypes with tumor promoting as well as repressing effects. Precise knowledge of the underlying interactions is the basis for a variety of treatment approaches, which are subsumed under the term antistromal therapy. Clinical implementation is still pending due to the lack of benefit-as well as paradoxical preclinical findings. While the prominent significance of CAFs in the immediate environment of the tumor is becoming clear, less is known about the circulating (c)CAFs. cCAFs are of particular interest as they seem not only to be potential new liquid biopsy biomarkers but also to support the survival of circulating tumor cells (CTC) in the bloodstream. In PDAC, CTCs correlate with an unfavorable outcome and can also be employed to monitor treatment response, but the current clinical relevance is limited. In this review, we discuss CTCs, cCAFs, secretomes that include EVs or fragments of collagen turnover as liquid biopsy biomarkers, and clinical approaches to target tumor stroma in PDAC.

Clinical applications and utility of cell-free DNA-based liquid biopsy analyses in cervical cancer and its precursor lesions.

Human papilloma virus (HPV) is an infectious carcinogenic agent. Nearly all cervical cancers are positive for one of the high-risk HPV subtypes. Although the introduction of the HPV vaccines in many countries have shown tremendous positive effects on the incidence of both cervical intraepithelial lesions (CIN) and invasive cancer, the large majority of females worldwide are still not vaccinated. Patients with diagnosed high-grade CIN need a lifelong close monitoring of possible relapse or development of invasive cancer. Different blood-based liquid biopsy approaches have shown great promise as an easily obtainable minimally invasive tool for early detection and monitoring of disease. Among the different liquid biopsy approaches the clinical relevance of cell-free DNA (cfDNA) in cervical cancer has been best investigated. In cervical cancer, the DNA fragments can be of both, human as well as viral origin. Thus, the mutation and methylation status of genes related to carcinogenesis as well as the HPV status can be analysed in plasma from cervical cancer patients. This review describes recent advances in different cfDNA approaches for early detection and monitoring of cervical cancer and its precursor lesions.

Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism.

Whole-brain radiotherapy (WBRT) is the treatment backbone for many patients with brain metastasis; however, its efficacy in preventing disease progression and the associated toxicity have questioned the clinical impact of this approach and emphasized the need for alternative treatments. Given the limited therapeutic options available for these patients and the poor understanding of the molecular mechanisms underlying the resistance of metastatic lesions to WBRT, we sought to uncover actionable targets and biomarkers that could help to refine patient selection. Through an unbiased analysis of experimental in vivo models of brain metastasis resistant to WBRT, we identified activation of the S100A9-RAGE-NF-κB-JunB pathway in brain metastases as a potential mediator of resistance in this organ. Targeting this pathway genetically or pharmacologically was sufficient to revert the WBRT resistance and increase therapeutic benefits in vivo at lower doses of radiation. In patients with primary melanoma, lung or breast adenocarcinoma developing brain metastasis, endogenous S100A9 levels in brain lesions correlated with clinical response to WBRT and underscored the potential of S100A9 levels in the blood as a noninvasive biomarker. Collectively, we provide a molecular framework to personalize WBRT and improve its efficacy through combination with a radiosensitizer that balances therapeutic benefit and toxicity.