Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy

Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the RARβ2 gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA.     

Oxidative Damage in Sporadic Colorectal Cancer: Molecular Mapping of Base Excision Repair Glycosylases MUTYH and hOGG1 in Colorectal Cancer Patients

Oxidative stress, oxidative DNA damage and resulting mutations play a role in colorectal carcinogenesis. Impaired equilibrium between DNA damage formation, antioxidant status, and DNA repair capacity is responsible for the accumulation of genetic mutations and genomic instability. The lesion-specific DNA glycosylases, e.g., hOGG1 and MUTYH, initiate the repair of oxidative DNA damage. Hereditary syndromes (MUTYH-associated polyposis, NTHL1-associated tumor syndrome) with germline mutations causing a loss-of-function in base excision repair glycosylases, serve as straight forward evidence on the role of oxidative DNA damage and its repair. Altered or inhibited function of above glycosylases result in an accumulation of oxidative DNA damage and contribute to the adenoma-adenocarcinoma transition. Oxidative DNA damage, unless repaired, often gives rise G:C > T:A mutations in tumor suppressor genes and proto-oncogenes with subsequent occurrence of chromosomal copy-neutral loss of heterozygosity. For instance, G>T transversions in position c.34 of a KRAS gene serves as a pre-screening tool for MUTYH-associated polyposis diagnosis. Since sporadic colorectal cancer represents more complex and heterogenous disease, the situation is more complicated. In the present study we focused on the roles of base excision repair glycosylases (hOGG1, MUTYH) in colorectal cancer patients by investigating tumor and adjacent mucosa tissues. Although we found downregulation of both glycosylases and significantly lower expression of hOGG1 in tumor tissues, accompanied with G>T mutations in KRAS gene, oxidative DNA damage and its repair cannot solely explain the onset of sporadic colorectal cancer. In this respect, other factors (especially microenvironment) per se or in combination with oxidative DNA damage warrant further attention. Base excision repair characteristics determined in colorectal cancer tissues and their association with disease prognosis have been discussed as well.     

Circulating Tumor DNA in Precision Oncology and Its Applications in Colorectal Cancer

Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that is shed by malignant tumors into the bloodstream and other bodily fluids. ctDNA can comprise up to 10% of a patient’s cfDNA depending on their tumor type and burden. The short half-life of ctDNA ensures that its detection captures tumor burden in real-time and offers a non-invasive method of repeatedly evaluating the genomic profile of a patient’s tumor. A challenge in ctDNA detection includes clonal hematopoiesis of indeterminate potential (CHIP), which can be distinguished from tumor variants using a paired whole-blood control. Most assays for ctDNA quantification rely on measurements of somatic variant allele frequency (VAF), which is a mutation-dependent method. Patients with certain types of solid tumors, including colorectal cancer (CRC), can have levels of cfDNA 50 times higher than healthy patients. ctDNA undergoes a precipitous drop shortly after tumor resection and therapy, and rising levels can foreshadow radiologic recurrence on the order of months. The amount of tumor bulk required for ctDNA detection is lower than that for computed tomography (CT) scan detection, with ctDNA detection preceding radiologic recurrence in many cases. cfDNA/ctDNA can be used for tumor molecular profiling to identify resistance mutations when tumor biopsy is not available, to detect minimal residual disease (MRD), to monitor therapy response, and for the detection of tumor relapse. Although ctDNA is not yet implemented in clinical practice, studies are ongoing to define the appropriate way to use it as a tool in the clinic. In this review article, we examine the general aspects of ctDNA, its status as a biomarker, and its role in the management of early (II–III) and late (IV; mCRC) stage colorectal cancer (CRC).     

Microsatellite Instability in Russian Patients with Colorectal Cancer

The aim of this study was to determine the characteristics of Russian patients with microsatellite instability (MSI) tumors. MSI in the tumor was determined in 514 patients with colon cancer using PCR and subsequent fragment analysis for five markers (NR21, NR24, BAT25, BAT26, and NR27). In the presence of microsatellite instability, the mismatch repair (MMR) system genes were examined using the NGS and MLPA methods to establish the diagnosis of Lynch syndrome. The overall frequency of MSI tumors was 15%: at stage I—19% (9/48), at stage II—21% (44/213), at stage III—16% (26/160), and at stage IV—2% (2/93). Patients with MSI tumors differed in the age of diagnosis, tumor localization, time of cancer recurrence, and stage of the disease. The overall and disease-free survival of patients whose tumors had MSI status was higher than that of patients with microsatellite-stable status, p = 0.04 and p = 0.02, respectively. Analysis of overall and disease-free survival of patients with Lynch syndrome and patients with sporadic colon cancer, but with MSI status, did not reveal significant differences, p = 0.52 and p = 0.24, respectively. The age of patients with Lynch syndrome was significantly younger than that of patients with sporadic colon cancer whose tumors had MSI status (p < 0.001).     

Inflammation-Related Biomarkers for the Prediction of Prognosis in Colorectal Cancer Patients

Colorectal cancer (CRC) is the leading cause of cancer deaths around the world. It is necessary to identify patients with poor prognosis or with high risk for recurrence so that we can selectively perform intensive treatments such as preoperative and/or postoperative chemotherapy and extended surgery. The clinical usefulness of inflammation-related prognostic biomarkers available from routine blood examination has been reported in many types of cancer, e.g., neutrophil–lymphocyte ratio (NLR), lymphocyte–C-reactive protein ratio (LCR), platelet–lymphocyte ratio (PLR), lymphocyte–monocyte ratio (LMR), and so on. Moreover, some scoring systems based on circulating blood cell counts and albumin concentration have been also reported to predict cancer patients’ prognosis, such as the Glasgow prognostic score (GPS), systemic inflammation score (SIS), and prognostic nutritional index (PNI). The optimal biomarker and optimal cutoff value of the markers can be different depending on the cancer type. In this review, we summarize the prognostic impact of each inflammation-related marker in CRC.     

Epigenetics Meets Radiation Biology as a New Approach in Cancer Treatment

Cancer is a disease that results from both genetic and epigenetic changes. In recent decades, a number of people have investigated the disparities in gene expression resulting from variable DNA methylation alteration and chromatin structure modification in response to the environment. Especially, colon cancer is a great model system for investigating the epigenetic mechanism for aberrant gene expression alteration. Ionizing radiation (IR) could affect a variety of processes within exposed cells and, in particular, cause changes in gene expression, disruption of cell cycle arrest, and apoptotic cell death. Even though there is growing evidence on the importance of epigenetics and biological processes induced by radiation exposure in various cancer types including colon cancer, specific epigenetic alterations induced by radiation at the molecular level are incompletely defined. This review focuses on discussing possible IR-mediated changes of DNA methylation and histone modification in cancer.     

The Homeobox Only Protein Homeobox (HOPX) and Colorectal Cancer

The HOP (homeobox only protein) homeobox (HOPX) is most closely related to the homeobox protein that contains a homeobox-like domain but lacks certain conserved residues required for DNA binding. Here, we review the current understanding of HOPX in the progression of colorectal cancer (CRC). HOPX was initially reported as a differentiation marker and is expressed in various normal tissues. In the colon, HOPX is expressed uniquely in the quiescent stem cell, +4, and in differentiated mucosal cells of the colon. HOPX expression is markedly suppressed in a subset of cancers, mainly in an epigenetic manner. CRC may include separate entities which are differentially characterized by HOPX expression from a prognostic point of view. HOPX itself can regulate epigenetics, and defective expression of HOPX can result in loss of tumor suppressive function and differentiation phenotype. These findings indicate that HOPX may be both a central regulator of epigenetic dynamics and a critical determinant for differentiation in human cells. HOPX downstream targets were identified in CRC cell lines and hold promise as candidates for therapeutic targets of CRC, such as EphA2 or AP-1. Further analysis will elucidate and confirm the precise role of such proteins in CRC progression.     

Lessons to Learn for Adequate Targeted Therapy Development in Metastatic Colorectal Cancer Patients

Insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase that regulates cell growth and proliferation. Upregulation of the IGF1R pathway constitutes a common paradigm shared with other receptor tyrosine kinases such as EGFR, HER2, and MET in different cancer types, including colon cancer. The main IGF1R signaling pathways are PI3K-AKT and MAPK-MEK. However, different processes, such as post-translational modification (SUMOylation), epithelial-to-mesenchymal transition (EMT), and microenvironment complexity, can also contribute to intrinsic and acquired resistance. Here, we discuss new strategies for adequate drug development in metastatic colorectal cancer patients.     

Biomarkers of Bladder Cancer: Cell-Free DNA,Epigenetic Modifications and Non-Coding RNAs

Bladder cancer (BC) is the 10th most frequent cancer in the world. The initial diagnosis and surveillance of BC require a combination of invasive and non-invasive methods, which are costly and suffer from several limitations. Cystoscopy with urine cytology and histological examination presents the standard diagnostic approach. Various biomarkers (e.g., proteins, genes, and RNAs) have been extensively studied in relation to BC. However, the new trend of liquid biopsy slowly proves to be almost equally effective. Cell-free DNA, non-coding RNA, and other subcellular structures are now being tested for the best predictive and diagnostic value. In this review, we focused on published gene mutations, especially in DNA fragments, but also epigenetic modifications, and non-coding RNA (ncRNA) molecules acquired by liquid biopsy. We performed an online search in PubMed/Medline, Scopus, and Web of Science databases using the terms “bladder cancer”, in combination with “markers” or “biomarkers” published until August 2022. If applicable, we set the sensitivity and specificity threshold to 80%. In the era of precision medicine, the development of complex laboratory techniques fuels the search and development of more sensitive and specific biomarkers for diagnosis, follow-up, and screening of BC. Future efforts will be focused on the validation of their sensitivity, specificity, predictive value, and their utility in everyday clinical practice.     

HOXB9 Overexpression Promotes Colorectal Cancer Progression and Is Associated with Worse Survival in Liver Resection Patients for Colorectal Liver Metastases

As is known, HOXB9 is an important factor affecting disease progression and overall survival (OS) in cancer. However, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the role of HOXB9 in CRC progression and its association with OS in colorectal liver metastases (CRLM). We analysed differential HOXB9 expression in CRC using the Tissue Cancer Genome Atlas database (TCGA). We modulated HOXB9 expression in vitro to assess its impact on cell proliferation and epithelial-mesenchymal transition (EMT). Lastly, we explored the association of HOXB9 protein expression with OS, using an institutional patient cohort (n = 110) who underwent liver resection for CRLM. Furthermore, HOXB9 was upregulated in TCGA-CRC (n = 644) vs. normal tissue (n = 51) and its expression levels were elevated in KRAS mutations (p < 0.0001). In vitro, HOXB9 overexpression increased cell proliferation (p < 0.001) and upregulated the mRNA expression of EMT markers (VIM, CDH2, ZEB1, ZEB2, SNAI1 and SNAI2) while downregulated CDH1, (p < 0.05 for all comparisons). Conversely, HOXB9 silencing disrupted cell growth (p < 0.0001). High HOXB9 expression (HR = 3.82, 95% CI: 1.59–9.2, p = 0.003) was independently associated with worse OS in CRLM-HOXB9-expressing patients after liver resection. In conclusion, HOXB9 may be associated with worse OS in CRLM and may promote CRC progression, whereas HOXB9 silencing may inhibit CRC growth.     

Colorectal Cancer Classification and Cell Heterogeneity: A Systems Oncology Approach

Colorectal cancer is a heterogeneous disease that manifests through diverse clinical scenarios. During many years, our knowledge about the variability of colorectal tumors was limited to the histopathological analysis from which generic classifications associated with different clinical expectations are derived. However, currently we are beginning to understand that under the intense pathological and clinical variability of these tumors there underlies strong genetic and biological heterogeneity. Thus, with the increasing available information of inter-tumor and intra-tumor heterogeneity, the classical pathological approach is being displaced in favor of novel molecular classifications. In the present article, we summarize the most relevant proposals of molecular classifications obtained from the analysis of colorectal tumors using powerful high throughput techniques and devices. We also discuss the role that cancer systems biology may play in the integration and interpretation of the high amount of data generated and the challenges to be addressed in the future development of precision oncology. In addition, we review the current state of implementation of these novel tools in the pathological laboratory and in clinical practice.     

Neutrophil Extracellular Traps in Colorectal Cancer Progression and Metastasis

Neutrophils form sticky web-like structures known as neutrophil extracellular traps (NETs) as part of innate immune response. NETs are decondensed extracellular chromatin filaments comprising nuclear and cytoplasmic proteins. NETs have been implicated in many gastrointestinal diseases including colorectal cancer (CRC). However, the regulatory mechanisms of NET formation and potential pharmacological inhibitors in the context of CRC have not been thoroughly discussed. In this review, we intend to highlight roles of NETs in CRC progression and metastasis as well as the potential of targeting NETs during colon cancer therapy.     

MicroRNA Methylome Signature and Their Functional Roles in Colorectal Cancer Diagnosis,Prognosis, and Chemoresistance

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Despite significant advances in the diagnostic services and patient care, several gaps remain to be addressed, from early detection, to identifying prognostic variables, effective treatment for the metastatic disease, and the implementation of tailored treatment strategies. MicroRNAs, the short non-coding RNA species, are deregulated in CRC and play a significant role in the occurrence and progression. Nevertheless, microRNA research has historically been based on expression levels to determine its biological significance. The exact mechanism underpinning microRNA deregulation in cancer has yet to be elucidated, but several studies have demonstrated that epigenetic mechanisms play important roles in the regulation of microRNA expression, particularly DNA methylation. However, the methylation profiles of microRNAs remain unknown in CRC patients. Methylation is the next major paradigm shift in cancer detection since large-scale epigenetic alterations are potentially better in identifying and classifying cancers at an earlier stage than somatic mutations. This review aims to provide insight into the current state of understanding of microRNA methylation in CRC. The new knowledge from this study can be utilized for personalized health diagnostics, disease prediction, and monitoring of treatment.     

Circulating Cell-Free Tumour DNA in the Management of Cancer

With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease.     

Granulin: An Invasive and Survival-Determining Marker in Colorectal Cancer Patients

Background: Granulin is a secreted, glycosylated peptide—originated by cleavage from a precursor protein—which is involved in cell growth, tumor invasion and angiogenesis. However, the specific prognostic impact of granulin in human colorectal cancer has only been studied to a limited extent. Thus, we wanted to assess the expression of granulin in colorectal cancer patients to evaluate its potential as a prognostic biomarker. Methods: Expressional differences of granulin in colorectal carcinoma tissue (n = 94) and corresponding healthy colon mucosa were assessed using qRT-PCR. Immunohistochemistry was performed in colorectal cancer specimens (n = 97), corresponding healthy mucosa (n = 47) and colorectal adenomas (n = 19). Subsequently, the results were correlated with histopathological and clinical patients’ data. HCT-116 cells were transfected with siRNA for invasion and migration assays. Results: Immunohistochemistry and qRT-PCR revealed tumoral over expression of granulin in colorectal cancer specimens compared to corresponding healthy colon mucosa and adenomas. Tumoral overexpression of granulin was associated with a significantly impaired overall survival. Moreover, downregulation of granulin by siRNA significantly diminished the invasive capacities of HCT-116 cells in vitro. Conclusion: Expression of granulin differs in colorectal cancer tissue, adenomas and healthy colon mucosa. Furthermore, granulin features invasive and migrative capabilities and overexpression of granulin correlates with a dismal prognosis. This reveals its potential as a prognostic biomarker and granulin could be a worthwhile molecular target for individualized anticancer therapy.     

The Therapeutic Potential of Milk Extracellular Vesicles on Colorectal Cancer

Colorectal cancer remains one of the leading prevalent cancers in the world and is the fourth most common cause of death from cancer. Unfortunately, the currently utilized chemotherapies fail in selectively targeting cancer cells and cause harm to healthy cells, which results in profound side effects. Researchers are focused on developing anti-cancer targeted medications, which is essential to making them safer, more effective, and more selective and to maximizing their therapeutic benefits. Milk-derived extracellular vesicles (EVs) from camels and cows have attracted much attention as a natural substitute product that effectively suppresses a wide range of tumor cells. This review sheds light on the biogenesis, methods of isolation, characterization, and molecular composition of milk EVs as well as the therapeutic potentials of milk EVs on colorectal cancer.     

Non-Invasive Profiling of Advanced Prostate Cancer via Multi-Parametric Liquid Biopsy and Radiomic Analysis

Integrating liquid biopsies of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) with other minimally invasive measures may yield more comprehensive disease profiles. We evaluated the feasibility of concurrent cellular and molecular analysis of CTCs and cfDNA combined with radiomic analysis of CT scans from patients with metastatic castration-resistant PC (mCRPC). CTCs from 22 patients were enumerated, stained for PC-relevant markers, and clustered based on morphometric and immunofluorescent features using machine learning. DNA from single CTCs, matched cfDNA, and buffy coats was sequenced using a targeted amplicon cancer hotspot panel. Radiomic analysis was performed on bone metastases identified on CT scans from the same patients. CTCs were detected in 77% of patients and clustered reproducibly. cfDNA sequencing had high sensitivity (98.8%) for germline variants compared to WBC. Shared and unique somatic variants in PC-related genes were detected in cfDNA in 45% of patients (MAF > 0.1%) and in CTCs in 92% of patients (MAF > 10%). Radiomic analysis identified a signature that strongly correlated with CTC count and plasma cfDNA level. Integration of cellular, molecular, and radiomic data in a multi-parametric approach is feasible, yielding complementary profiles that may enable more comprehensive non-invasive disease modeling and prediction.     

The Immunotherapy for Colorectal Cancer,Lung Cancer and Pancreatic Cancer

Immunotherapy is a novel anti-cancer method which employs a different mechanism to conventional treatment. It has become a significant strategy because it provides a better or an alternative option for cancer patients. Recently, immunotherapy has been increasingly approved for the treatment of cancer; however, it has various limitations; for instance, it is only suitable for specific patients, the response rate is still low in most cases, etc. Colorectal cancer, lung cancer and pancreatic cancer are known as three major death-causing cancers in most countries. In this review, we discuss immunotherapeutic treatment for these three cancers, and consider the option, prospects and limitations of immunotherapy. The development of immunotherapy should focus on the discovery of biomarkers to screen suitable patients, new targets on tumors, neoadjuvant immunotherapy and the combination of immunotherapy with conventional therapeutic methods. We can expect that immunotherapy potentially will develop as one of the best therapies for patients with advanced cancer or poor responses to traditional methods.     

Circulating miRNA Expression Profiles and Machine Learning Models in Association with Response to Irinotecan-Based Treatment in Metastatic Colorectal Cancer

Colorectal cancer represents a leading cause of cancer-related morbidity and mortality. Despite improvements, chemotherapy remains the backbone of colorectal cancer treatment. The aim of this study is to investigate the variation of circulating microRNA expression profiles and the response to irinotecan-based treatment in metastatic colorectal cancer and to identify relevant target genes and molecular functions. Serum samples from 95 metastatic colorectal cancer patients were analyzed. The microRNA expression was tested with a NucleoSpin miRNA kit (Machnery-Nagel, Germany), and a machine learning approach was subsequently applied for microRNA profiling. The top 10 upregulated microRNAs in the non-responders group were hsa-miR-181b-5p, hsa-miR-10b-5p, hsa-let-7f-5p, hsa-miR-181a-5p, hsa-miR-181d-5p, hsa-miR-301a-3p, hsa-miR-92a-3p, hsa-miR-155-5p, hsa-miR-30c-5p, and hsa-let-7i-5p. Similarly, the top 10 downregulated microRNAs were hsa-let-7d-5p, hsa-let-7c-5p, hsa-miR-215-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-miR-10a-5p, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-122-5p, and hsa-miR-17-5p. The upregulation of microRNAs in the miR-181 family and the downregulation of those in the let-7 family appear to be mostly involved with non-responsiveness to irinotecan-based treatment.