Predictive Significance of Combined Plasmatic Detection of BRAF Mutations and S100B Tumor Marker in Early-Stage Malignant Melanoma.

BACKGROUND: Melanoma is the most aggressive skin cancer with ability to recur also after early-stage tumor surgery. The aim was to identify early-stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. METHODS: Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre-amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. RESULTS: The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild-type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). CONCLUSION: We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. SIGNIFICANCE: There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early-stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre- and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage.

Prognostic Value of Tumor Tissue Up-regulated microRNAs in Clear Cell Renal Cell Carcinoma (ccRCC).

BACKGROUND/AIM: The management of patients with clear cell renal cell carcinoma (ccRCC) includes prognosis assessment based on TNM classification and biochemical markers. This approach stratifies patients with advanced ccRCC into groups of favorable, intermediate, and poor prognosis. The aim of the study was to improve prognosis estimation using microRNAs involved in the pathogenesis of ccRCC. PATIENTS AND METHODS: The study was based on a histologically-verified set of matched ccRCC FFPE tissue samples (normal renal tissue, primary tumor, metastasis, n=20+20+20). The expression of 2,549 microRNAs was analyzed using the SurePrint G3 Human miRNA microarray kit (Agilent Technologies). Prognostic value of significantly deregulated microRNAs was further evaluated on microRNA expression and clinical data of 475 patients obtained from TCGA Kidney Clear Cell Carcinoma (KIRC) database. RESULTS: There were 13 up-regulated and 6 down-regulated microRNAs in tumor tissues compared to control tissues. Among them, survival analysis revealed those with prognostic significance. Patients with high expression of miR-21, miR-27a, miR-34a, miR-106b, miR-210, and miR-342 showed significantly unfavorable outcome. The opposite was observed for miR-30e, patients with low expression had significantly shorter survival. CONCLUSION: The inclusion of these microRNAs in a prognostic panel holds the potential to enhance stratification scoring systems, on which the treatment of ccRCC patients is based.

BRAF mutation, selected miRNAs and genes expression in primary papillary thyroid carcinomas and local lymph node metastases.

Mutations in cancer-related genes are now known to be accompanied by epigenetic events in carcinogenesis by modification of the regulatory pathways and expression of genes involved in the pathobiology. Such cancer-related mutations, miRNAs and gene expression may be promising molecular markers of the most common papillary thyroid carcinoma (PTC). However, there are limited data on their relationships. The aim of this study was to analyse the interactions between BRAF mutations, selected microRNAs (miR-21, miR-34a, miR-146b, and miR-9) and the expression of selected genes (LGALS3, NKX2-1, TACSTD2, TPO) involved in the pathogenesis of PTC. The study cohort included 60 primary papillary thyroid carcinomas (PTC) that were classified as classical (PTC/C; n=50) and invasive follicular variant (PTC/F; n=10), and 40 paired lymph node metastases (LNM). BRAF mutation status in primary and recurrent/persistent papillary thyroid carcinomas was determined. The mutation results were compared both between primary and metastatic cancer tissue, and between BRAF mutation status and selected genes and miRNA expression in primary PTC. Furthermore, miRNAs and gene expression were compared between primary PTCs and non-neoplastic tissue, and local lymph node metastatic tumor, respectively. All studied markers showed several significant mutual interactions and contexts. In conclusion, to the best our knowledge, this is the first integrated study of BRAF mutational status, the expression levels of mRNAs of selected genes and miRNAs in primary PTC, and paired LNM.

Next Generation Sequencing Analysis and its Benefit for Targeted Therapy of Lung Adenocarcinoma.

BACKGROUND/AIM: Targeted therapy has become increasingly important in treating lung adenocarcinoma, the most common subtype of lung cancer. Next-generation sequencing (NGS) enables precise identification of specific genetic alterations in individual tumor tissues, thereby guiding targeted therapy selection. This study aimed to analyze mutations present in adenocarcinoma tissues using NGS, assess the benefit of targeted therapy and evaluate the progress in availability of targeted therapies over last five years. PATIENTS AND METHODS: The study included 237 lung adenocarcinoma patients treated between 2018-2020. The Archer FusionPlex CTL panel was used for NGS analysis. RESULTS: Gene variants covered by the panel were detected in 57% patients and fusion genes in 5.9% patients. At the time of the study, 34 patients (14.3% of patients) were identified with a targetable variant. Twenty-five patients with EGFR variants, 8 patients with EML4-ALK fusion and one patient with CD74-ROS1 fusion received targeted therapy. Prognosis of patients at advanced stages with EGFR variants treated by tyrosine kinase inhibitors and patients with EML4-ALK fusion treated by alectinib was significantly favorable compared to patients without any targetable variant treated by chemotherapy (p=0.0172, p=0.0096, respectively). Based on treatment guidelines applicable in May 2023, the number of patients who could profit from targeted therapy would be 64 (27.0% of patients), this is an increase by 88% in comparison to recommendations valid in 2018-2020. CONCLUSION: As lung adenocarcinoma patients significantly benefit from targeted therapy, the assessment of mutational profiles using NGS could become a crucial approach in the routine management of oncological patients.

Preoperative Plasma miRNA Levels Predict Prognosis in Early-stage Malignant Melanoma.

BACKGROUND/AIM: Non-invasive circulating tumor biomarkers in liquid biopsy, such as microRNAs (miRNA), provide for better personalization of treatment strategies. The aim of our study was to assess the prognosis of patients with melanoma undergoing tumor resection with curative intent based on analysis of selected circulating miRNAs. PATIENTS AND METHODS: A total of 22 patients with stage I to III melanoma were enrolled into this prospective study. Plasma samples were obtained pre-surgery and early post-surgery from peripheral blood draws. A panel of 23 candidate miRNAs was designed and expression of miRNAs were analyzed by reverse transcription-quantitative polymerase chain reaction with exogenous reference control cel-miR-39-3p. RESULTS: Higher preoperative expression levels of miR-99a (p=0.008), miR-320 (p=0.009), miR-1908 (p=0.001), miR-494 (p=0.018) and miR-4487 (p=0.048) were associated with a shorter disease-free interval. Similarly, higher preoperative plasma levels of miR-99a (p=0.017), miR-221 (p=0.026), miR-320 (p=0.016), miR-494 (p=0.009), miR-1260 (p=0.026) and miR-1908 (p=0.024) were associated with worse overall survival. No significant differences between pre- and postoperative plasma miRNA levels were observed. CONCLUSION: Liquid biopsy is a minimally-invasive approach which can lead to a better understanding of cancer behavior and offers the possibility of precise patient prognosis, allowing selection of the most appropriate treatment. Our study showed that preoperative plasma levels of miR-99a, miR-221, miR-320, miR-494, miR-1908 and miR-4487 were associated with disease-free interval and overall survival of patients with early-stage melanoma. This approach may help in decision-making about the appropriateness of modern adjuvant treatment administration in patients with resectable melanoma.

Applications of Liquid Biopsies in Non-Small-Cell Lung Cancer.

The concept of liquid biopsy as an analysis tool for non-solid tissue carried out for the purpose of providing information about solid tumors was introduced approximately 20 years ago. Additional to the detection of circulating tumor cells (CTCs), the liquid biopsy approach quickly included the analysis of circulating tumor DNA (ctDNA) and other tumor-derived markers such as circulating cell-free RNA or extracellular vesicles. Liquid biopsy is a non-invasive technique for detecting multiple cancer-associated biomarkers that is easy to obtain and can reflect the characteristics of the entire tumor mass. Currently, ctDNA is the key component of the liquid biopsy approach from the point of view of the prognosis assessment, prediction, and monitoring of the treatment of non-small-cell lung cancer (NSCLC) patients. ctDNA in NSCLC patients carries variants or rearrangements that drive carcinogenesis, such as those in EGFR, KRAS, ALK, or ROS1. Due to advances in pharmacology, these variants are the subject of targeted therapy. Therefore, the detection of these variants has gained attention in clinical medicine. Recently, methods based on qPCR (ddPCR, BEAMing) and next-generation sequencing (NGS) are the most effective approaches for ctDNA analysis. This review addresses various aspects of the use of liquid biopsy with an emphasis on ctDNA as a biomarker in NSCLC patients.

BAI1 as a Prognostic Marker of Clear Cell Renal Cell Carcinoma (ccRCC).

BACKGROUND/AIM: The treatment of advanced clear cell renal cell carcinoma (ccRCC) is based on stratification of patients according to prognosis (favorable, intermediate, and poor). The aim of the study was to improve prognostication by biomarkers involved in angiogenesis. PATIENTS AND METHODS: The study group consisted of 20 patients who underwent surgery for ccRCC. Gene expression analysis was peformed on a set of matched (primary tumor, metastasis, n=20+20) FFPE tissue samples. An additional analysis was done on expression data of 606 patients obtained from the TCGA Kidney Clear Cell Carcinoma (KIRC) database. Quantitative estimation of mRNA of selected genes (TaqMan human Angiogenesis Array, 97 genes) was performed by a real-time RT-PCR method with TaqMan® arrays. RESULTS: Using the Cox regression model, 4 genes (PDGFB, FGF4, EPHB2 and BAI1) were identified whose expression was related to progression-free interval (PFI). Further analysis using the Kaplan Meier method conclusively revealed the relationship of BAI1 expression to prognosis (both datasets). Patients with higher BAI1 expression had significantly shorter PFI and overall survival. CONCLUSION: We showed that tumor tissue BAI1 expression level is a prognostic marker in ccRCC. Therefore, this gene might be involved in a prognostic panel to improve scoring systems on which the management of metastatic ccRCC patients is based.

The potential of miR-499 plasmatic level as a biomarker of obstructive sleep apnea syndrome.

Background: Obstructive sleep apnea syndrome (OSAS) is one of the most common sleep-related breathing disorders. The aim of this study was to improve diagnostics in OSAS using blood circulating biomarkers. We consider the potential of cardiac-specific miRNAs in the diagnosis and risk assessment of cardiovascular complications. Materials & methods: Plasmatic levels of miR-1-3p, miR-133a-3p and miR-499a-5p were measured by reverse transcription-PCR and compared with the clinical status of OSAS patients and controls. Results: The level of miR-499 was higher (p = 0.0343) in OSAS patients (mean expression: 0.00561) compared with the controls (mean expression: 0.00003), using the multivariate logistic regression. Conclusion: The role of miR-499 in gene expression regulation during hypoxia and our findings indicate that miR-499 could be a new diagnostic biomarker for OSAS.

Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model.

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.

Prognostic Significance of Lymph Node Examination by the OSNA Method in Lung Cancer Patients-Comparison with the Standard Histopathological Procedure.

The aim of the study was to compare the prognostic significance of lymph node status of patients with lung cancer analyzed by three different methods: hematoxylin and eosin (H&E), immunohistochemistry of cytokeratin 19 (IHC CK19), and One-Step Nucleic Acid Amplification (OSNA). The clinical relevance of the results was evaluated based on relation to prognosis; the disease-free interval (DFI) and overall survival (OS) were analyzed. During radical surgical treatment, a total of 1426 lymph nodes were obtained from 100 patients, creating 472 groups of nodes (4-5 groups per patient) and examined by H&E, IHC CK19 and OSNA. The median follow-up was 44 months. Concordant results on the lymph node status of the H&E, IHC CK19 and OSNA examinations were reported in 78% of patients. We recorded shorter OS in patients with positive results provided by both OSNA and H&E. The study demonstrated a higher percentage of detected micrometastases in lymph nodes by the OSNA method. However, the higher sensitivity of the OSNA, with the cut-off value 250 copies of mRNA of CK19/µL, resulted in a lower association of OSNA positivity with progress of the disease compared to H&E. Increasing the cut-off to 615 copies resulted in an increase in concordance between the OSNA and H&E, which means that the higher cut-off is more relevant in the case of lung tumors.

The Level of Preoperative Plasma KRAS Mutations and CEA Predict Survival of Patients Undergoing Surgery for Colorectal Cancer Liver Metastases.

Colorectal cancer (CRC) belongs to the most common cancers. The liver is a predominant site of CRC dissemination. Novel biomarkers for predicting the survival of CRC patients with liver metastases (CLM) undergoing metastasectomy are needed. We examined KRAS mutated circulating cell-free tumor DNA (ctDNA) in CLM patients as a prognostic biomarker, independently or in combination with carcinoembryonic antigen (CEA). Thereby, a total of 71 CLM were retrospectively analyzed. Seven KRAS G12/G13 mutations was analyzed by a ddPCR™ KRAS G12/G13 Screening Kit on QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA, USA) in liver metastasis tissue and preoperative and postoperative plasma samples. CEA were determined by an ACCESS CEA assay with the UniCel DxI 800 Instrument (Beckman Coulter, Brea, CA, USA). Tissue KRAS positive liver metastases was detected in 33 of 69 patients (47.8%). Preoperative plasma samples were available in 30 patients and 11 (36.7%) were KRAS positive. The agreement between plasma- and tissue-based KRAS mutation status was 75.9% (22 in 29; kappa 0.529). Patients with high compared to low levels of preoperative plasma KRAS fractional abundance (cut-off 3.33%) experienced shorter overall survival (OS 647 vs. 1392 days, p = 0.003). The combination of high preoperative KRAS fractional abundance and high CEA (cut-off 3.33% and 4.9 µg/L, resp.) best predicted shorter OS (HR 13.638, 95%CI 1.567-118.725) in multivariate analysis also (OS HR 44.877, 95%CI 1.59-1266.479; covariates: extend of liver resection, biological treatment). KRAS mutations are detectable and quantifiable in preoperative plasma cell-free DNA, incompletely overlapping with tissue biopsy. KRAS mutated ctDNA is a prognostic factor for CLM patients undergoing liver metastasectomy. The best prognostic value can be reached by a combination of ctDNA and tumor marker CEA.

Plasma microRNA Levels Combined with CEA and CA19-9 in the Follow-Up of Colorectal Cancer Patients.

Colorectal cancer (CRC) ranks among the most common cancers worldwide. Surgical removal remains the best strategy for treatment of resectable tumors. An important part of caring for patients after surgery is monitoring for early detection of a possible relapse of the disease. Efforts are being made to improve the sensitivity and specificity of routinely used carcinoembryonic antigen (CEA) with the use of additional biomarkers such as microRNAs. The aim of our study was to evaluate the prognostic potential of microRNAs and their use as markers of disease recurrence. The quantitative estimation of CEA, CA19-9, and 22 selected microRNAs (TaqMan Advanced miRNA Assays) was performed in 85 paired (preoperative and postoperative) blood plasma samples of CRC patients and in samples taken during the follow-up period. We have revealed a statistically significant decrease in plasma levels for miR-20a, miR-23a, miR-210, and miR-223a (p = 0.0093, p = 0.0013, p = 0.0392, and p = 0.0214, respectively) after surgical removal of the tumor tissue. A statistically significant relation to prognosis (overall survival; OS) was recorded for preoperative plasma levels of miR-20a, miR-21, and miR-23a (p = 0.0236, p = 0.0316, and p =0.0271, respectively) in a subgroup of patients who underwent palliative surgery. The best discrimination between patients with favorable and unfavorable outcomes was achieved by a combination of CEA, CA19-9 with miR-21, miR-20a, and miR-23a (p < 0.0001). The use of these microRNAs for early disease recurrence detection was affected by a low specificity in comparison with CEA and CA19-9. CEA and CA19-9 had high specificity but low sensitivity. Our results show the benefit of combining currently used standard biomarkers and microRNAs for precise prognosis estimation.

Sarcoma Stem Cell Heterogeneity.

Sarcomas represent an extensive group of divergent malignant diseases, with the only common characteristic of being derived from mesenchymal cells. As such, sarcomas are by definition very heterogeneous, and this heterogeneity does not manifest only upon intertumoral comparison on a bulk tumor level but can be extended to intratumoral level. Whereas part of this intratumoral heterogeneity could be understood in terms of clonal genetic evolution, an essential part includes a hierarchical relationship between sarcoma cells, governed by both genetic and epigenetic influences, signals that sarcoma cells are exposed to, and intrinsic developmental programs derived from sarcoma cells of origin. The notion of this functional hierarchy operating within each tumor implies the existence of sarcoma stem cells, which may originate from mesenchymal stem cells, and indeed, mesenchymal stem cells have been used to establish several crucial experimental sarcoma models and to trace down their respective stem cell populations. Mesenchymal stem cells themselves are heterogeneous, and, moreover, there are alternative possibilities for sarcoma cells of origin, like neural crest-derived stem cells, or mesenchymal committed precursor cells, or - in rhabdomyosarcoma - muscle satellite cells. These various origins result in substantial heterogeneity in possible sarcoma initiation. Genetic and epigenetic changes associated with sarcomagenesis profoundly impact the biology of sarcoma stem cells. For pediatric sarcomas featuring discrete reciprocal translocations and largely stable karyotypes, the translocation-activated oncogenes could be crucial factors that confer stemness, principally by modifying transcriptome and interfering with normal epigenetic regulation; the most extensively studied examples of this process are myxoid/round cell liposarcoma, Ewing sarcoma, and synovial sarcoma. For adult sarcomas, which have typically complex and unstable karyotypes, stemness might be defined more operationally, as a reflection of actual assembly of genetically and epigenetically conditioned stemness factors, with dedifferentiated liposarcoma providing a most thoroughly studied example. Alternatively, stemness can be imposed by tumor microenvironment, as extensively documented in osteosarcoma. In spite of this heterogeneity in both sarcoma initiation and underlying stemness biology, some of the molecular mechanisms of stemness might be remarkably similar in diverse sarcoma types, like abrogation of classical tumor suppressors pRb and p53, activation of Sox-2, or inhibition of canonical Wnt/ß-catenin signaling. Moreover, even some stem cell markers initially characterized for their stem cell enrichment capacity in various carcinomas or leukemias seem to function quite similarly in various sarcomas. Understanding the biology of sarcoma stem cells could significantly improve sarcoma patient clinical care, leading to both better patient stratification and, hopefully, development of more effective therapeutic options.

One-step nucleic acid amplification vs ultrastaging in the detection of sentinel lymph node metastasis in endometrial cancer patients.

BACKGROUND AND OBJECTIVES: Utilisation of the one-step nucleic acid amplification (OSNA) molecular biology method for the detection of the metastatic involvement of sentinel lymph nodes (SLNs) in endometrial cancer (EC) patients. A comparison with histopathological ultrastaging and a description of the clinical consequences. METHODS: Surgically treated EC patients underwent detection of SLNs. Nodes greater than 5 mm were cut into sections 2-mm thick parallel to the short axis of the node. Odd sections were examined according to the OSNA method, while even ones according to an appropriate ultrastaging protocol. Nodes less than or equal to 5 mm were cut into halves along the longitudinal axis with one half examined according to the OSNA method and the other half by ultrastaging. RESULTS: Fifty-eight patients were included and 135 SLNs were acquired. Both ultrastaging and OSNA agreed on 116 results. According to the OSNA method, 20.69% more patients were classified into International Federation of Gynecology and Obstetrics (FIGO) stage III. When comparing the results of the OSNA method to the conclusions of ultrastaging as a reference method, sensitivity of 90.9%, specificity of 85.5% and concordance of 85.9% were attained. CONCLUSIONS: The results of the OSNA method showed a higher frequency of detection of micrometastases and included 20.69% more patients into FIGO stage III.

Predictive relevance of miR-34a, miR-224 and miR-342 in patients with advanced squamous cell carcinoma of the lung undergoing palliative chemotherapy.

Attributing to their pathophysiological role and stability in biological samples, microRNAs (miRNAs) have the potential to become valuable predictive markers for non-small cell lung cancer (NSCLC). Samples of biopsy tissue constitute suitable material for miRNA profiling with the aim of predicting the effect of palliative chemotherapy. The present study group included 81 patients (74 males, 7 females, all smokers or former smokers) with the squamous cell carcinoma (SCC) histological subtype of NSCLC at a late stage (3B or 4). All patients received palliative chemotherapy based on platinum derivatives in combination with paclitaxel or gemcitabine. The expression of 17 selected miRNAs was measured by reverse transcription-quantitative polymerase chain reaction in tumor tissue macrodissected from formalin-fixed paraffin-embedded (FFPE) tissue samples. To predict the effect of palliative chemotherapy, the association between gene expression levels and overall survival (OS) time was analyzed. From the 17 miRNAs of interest, low expression levels of miR-342 and high expression levels of miR-34a and miR-224 were associated with a reduced OS time in subgroups of patients based on smoking status and treatment modality. Using cluster analysis, associations between combinations of miR-34a, -224 and -342 expression levels with patient survival were identified. The present study revealed that patients with the simultaneous high expression of miR-224 and -342 had a similar prognostic outcome to those with the low expression of miR-224 and -342, which was significantly reduced, compared with patients exhibiting high expression of either miR-224 or miR-342 with low expression of the other. We hypothesize that the effect of a particular miRNA is dependent on the expression level of other members of the miRNA network. This finding appears to complicate survival analyses based on individual miRNAs as markers. In conclusion, the present study provides evidence that specific miRNAs were associated with OS time, which may be candidate predictors for the effectiveness of palliative treatment in SCC lung cancer patients. This objective can be better achieved by combining more markers together than by using individual miRNAs.