Vol 52, No 3 (2024)
ARTICLES
The content of soluble forms of galectins -1, -3, -4, -7, -9 in patients with renal cell cancer of various morphological types
Abstract
Background: Galectins are a family of β-galactoside binding proteins that regulate the vast majority of cellular functions, including proliferation, migration, adhesion, and phagocytosis in both health and disease. More and more experimental and clinical evidence indicates that galectins are involved in many stages of carcinogenesis, including patients with renal cell carcinoma (RCC).
Aim: To analyze the clinical significance of soluble forms of galectins -1, -3, -4, -7, -9 in patients with various histological RCC types.
Materials and methods: We performed a retrospective analysis of the clinical significance of galectins -1, -3, -4, -7, -9 in the serum of 140 RCC patients (84 with clear cell RCC (ccRCC), 38 with papillary (papRCC), 18 with chromophobe (chrRCC)) and in 73 healthy donors (control group), who were examined and treated from 2019 to 2023 in the N. N. Blokhin National Medical Research Center of Oncology. Galectin levels were measured in serum (obtained according to standard methods before the initiation of specific treatment) with an enzyme-linked immunosorbent assay.
Results: There was a significant increase in serum galectin -1, -3, -9 levels in the whole RCC patient group, compared to the healthy donor control group; no increase was found for galectins -4 and -7. Serum galectin-1 levels in the ccRCC and papRCC patients were significantly higher than those in the controls (p = 0.0003 and p = 0.0135, respectively). No association between the serum galectins -1 and -7 and the clinical and morphological characteristics of RCC was found; however, serum galectin-7 levels in the papRCC patients correlated with the grade of tumor differentiation (r = -0.592; p = 0.001). The area under the ROC curve (AUC) for galectin-1 in ccRCC was 0.721 (p < 0.0001), in papRCC 0.673 (p = 0.0086), and in chrRCC 0.576 (p = 0.355). For galectin-7, the ROC AUC values were 0.527 (p = 0.634) in ccRCC, 0.513 (p = 0.845) in papRCC, and 0.566 (p = 0.425) in chrRCC. In all histological types of RCC, there was a significant increase in serum galectin-3 compared to the controls (ccRCC, p = 0.0208; papRCC, p = 0.0014; chrRCC, p = 0.0041). The ROC analysis for galectin-3 in patients with RCC of various histological types showed AUC = 0.721 (p < 0.0001) for ccRCC, 0.673 (p = 0.0086) for papRCC, and 0.576 (p = 0.355) for chrRCC. Galectin-9 levels was directly and significantly associated with the tumor size, as well as with regional metastases (r = 0.251, p = 0.021; r = 0.239, p = 0.028, respectively). The AUC values for galectin-4 were 0.619 (p = 0.021) in ccRCC, 0.577 (p = 0.214) in papRCC, and 0.534 (p = 0.666) for chrRCC. For galectin-9, they were 0.649 (p = 0.0075), 0.613 (p = 0.087), and 0.539 (p = 0.637), respectively.
Conclusion: The study has demonstrated a certain association between serum galectin -1, -3, -4, -7, and -9 in the patients with RCC of various histological types. Although the results of the ROC analysis indicated average quality of the model, which does not allow for the use of the obtained data for diagnostic purposes, it is necessary to continue the research for better understanding of the mechanisms of galectin functioning, before galectin-based therapeutic agents would be introduced into clinical practice for the treatment of RCC.
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Detection of colorectal cancer associated circular RNAs hsa_circ_0031263, hsa_circ_0072715, and hsa_circ_0136666 in plasma with nanowire chips
Abstract
Rationale: Colorectal cancer (CRC) is one of the most prevalent oncological diseases with high mortality. Invasive optical (endoscopic) colono- scopy has been recognized as a golden standard for the CRC diagnostics. A promising area is the development of non-invasive tools for CRC diagnosis with circular RNA (circRNA). One of the most sensitive non-invasive tools for detection of cancer RNA markers is considered to be the biosensor methods with the use of nanowire chips with oDNA probes (fragments of DNA oligonucleotides) immobilized on their surface. It has been previously shown that circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1 are associated with CRC.
Aim: To determine the lower limit of concentration sensitivity of detection of CRC-associated circRNA with nanowire chips with immobilized oDNA probes, to demonstrate the usability of these chips for non-invasive detection of circRNA in plasma in the CRC diagnostics, and to establish the potential for the use of nanowire chips for the early CRC diagnosis.
Methods: To ensure biospecific binding of the circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1 (the CRC markers), oDNA probes with the nucleotide sequences complementary to the target circRNA have been immobilized on the nanowire surface. At the study step 1, we detected the lower concentration limit for detection of the target molecules with the use of their analogues, i.e. synthetic model oDNA with the nucleotide sequences complementary to oDNA probes. At the study step 2, we used the nanowire chips with immobilized oDNA probes to detect the circRNA in plasma of the patients with confirmed CRC. Plasma samples from non-cancer patients were used as controls.
Results: The lower concentration limit for the detection of DNA analogues of the circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1 with nanowire chips with oDNA probes was 10-16 М. The analysis of total RNA isolated from plasma of the CRC patients showed a significant increase in the signal from the sensory elements of the nanowire chip. The analysis of plasma samples from the non-cancer patients, the nanowire signal changes were non-significant indicating the absence of detectable concentrations of the circRNA in plasma of the non-cancer patients.
Conclusion: We have identified the minimal detectable concentration of the circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1, associated to the development of CRC, with nanowire chips with immobilized oDNA probes: it was 10-16 М. The experiment showed the usability of such nanowire chips for non-invasive detection of the given circRNA markers in total RNA samples isolated from plasma of CRC patients.
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Identification of activating somatic mutations in the PIK3CA gene in breast tumors and determination of their minimal set for clinical diagnostic testing
Abstract
Background: For effective screening of breast cancer patients for candidates for target therapy with alpelisib, it is necessary to identify activating somatic mutations in the PIK3CA gene by allele specific polymerase chain reaction (PCR); this requires that an optimal list of mutations should be compiled.
Aim: To determine the spectrum of somatic mutations in the PIK3CA gene in breast cancer tumors by means of high performance sequencing (next generation sequencing, NGS) and to identify their minimal set for clinical diagnostic testing by allele specific PCR.
Methods: Targeted NGS was used to identify mutations in the PIK3CA gene in DNA obtained from paraffin blocks with tumor material from 431 patients with HR+HER2- breast cancer. A set of the most common somatic mutations was also detected by allele specific PCR.
Results: We have developed a set of reagents and a protocol for targeted NGS of frequently mutating regions of the PIK3CA and ESR1 genes, which was used to analyze samples from 451 HR+/HER2- breast cancer patients. Clinically significant activating mutations in the PIK3CA gene were found in 32.7% of the samples (141/431). The frequency of the mutant allele ranged from 0.15 to 0.65. Six mutations were most common: c.3140A>G p.His1047Arg (69), c.1633G>A p.Glu545Lys (32), c.1035T>A p.Asn345Lys (12), c.1624G>A p.Glu542Lys (9), c.3140A>T p.His1047Leu (8), Cys420Arg c.1258T>C (3). In total, these mutations amounted to 94.3% (133/141). In 3.5% of the samples (15/431), there were clinically significant somatic mutations in the ESR1 gene: c.1613A>G p, Asp538Gly (7), c.1610A>C p.Tyr537Ser (6), c.1609T>A p.Tyr537Asn (1), c.1610A>G p.Tyr537Cys (1), causing resistance to hormone therapy in patients with breast cancer. While rare mutations comprised only 5.7% of our sample, we validated a set of reagents to identify the six mutations described above by allele specific PCR. NGS and PCR were completely concordant.
Conclusion: PCR testing of activating somatic mutations of the PIK3CA gene meets the requirements for sensitivity (> 90%) and specificity (100%) for a clinical test and can be used in the selection of patients for targeted therapy with PIK3CA inhibitors.
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Association between full blood count and urine steroid metabolome in patients with adrenal masses
Abstract
Background: Endogenous hypercortisolism of adrenal origin is commonly associated with immune suppression. However, these patients also have signs characteristic of chronic inflammatory diseases. Better understanding of the mechanisms that alter the functioning of the immune system would allow for the development of a patient-centered approach to the treatment of corticotropin-independent endogenous Cushing's syndrome (CS).
Aim: To assess the association between full blood count and gas chromatography-mass spectrometry-based urinary steroid excretion in patients with adrenal masses depending on malignancy grade and presence of hypercortisolism.
Materials and methods: We retrospectively analyzed data from 42 patients with adrenal masses who had not received chemotherapy. The median age of the patients was 54 [Q25; Q75: 37; 63] years, and 76% of them were female. Preoperatively, all patients had hematology tests with differential leukocyte count. Steroid metabolome was assessed with Shimadzu GCMS-TQ8050 gas chromatography-mass spectrometer.
Results: Twelve (12) patients had adrenocortical cancer (ACC) and CS, 9 patients had ACC without CS, 11 had adrenocortical adenomas (ACA) and CS, and 10 patients had ACA without CS. ACC patients had a higher neutrophil-to-lymphocyte ratio (NLR) than those with ACA: 3.35 [2.5; 6.3] vs 1.99 [1.41; 2.65] (р = 0.001). There was a linear correlation between NLR and serum cortisol levels after the 1 mg overnight dexamethasone suppression test (r = 0.41, p = 0.01), urinary excretion of 5β-tetrahydrocortisol (5β-THF) (r = 0.71, p < 0.001) and 11β-hydroxyandrosterone (11β-OH-An) (r = 0.74, p < 0.001). The ACC patients without CS had lower 5β-THF urinary excretion values, compared to ACA with CS patients: 931 [616; 1610] and 3139 [1480; 4375] mcg/24h, respectively (р = 0.007). 11β-OH-An urinary excretion in ACC patients without CS was higher than in ACA patients with CS: 1170 [806; 1266] и 602 [320; 739] mcg/24h (р = 0.007). The NLR cut-off value for adrenal mass malignancy in patients with CS exceeded 2.72 (sensitivity 90.0%, specificity 80.0%), and for the patients without hypercortisolism was above 1.92 (sensitivity 71.4%, specificity 100.0%).
Conclusion: This is the first association identification between NLR, which is the marker of systemic inflammation, inflammation, and urinary excretion of 11β-OH-An, a metabolite of 11-hydroxyandrostenedione (a member of 11-oxygenated androgen family). This extends our understanding of the impact of hormonal activity of adrenal mass cells on the immune system.
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The group of hypermethylated long noncoding RNA genes is associated with different types of metastasis in ovarian cancer
Abstract
Background: Ovarian tumors are characterized by asymptomatic progression until their late stages, when at the time of diagnosis the patient already has extensive metastatic disease. In addition to lymphogenous and hematogenous metastasis in ovarian cancer, there are peritoneal dissemination and metastasis to the greater omentum with ascites; moreover, peritoneal carcinomatosis is the predominant route of metastasizing of ovarian cancer. Epigenetic factors, such as gene methylation, regulatory microRNAs and long non-coding RNAs (lncRNAs), contribute to progression of this cancer. Our previous bioinformatic and experimental studies have identified 13 genes of lncRNAs (GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, PLUT/PDX1-AS1, SNHG1, SNHG6, SNHG12, SNHG17, TINCR, TP53TG1, TUG1) hypermethylated in the ovarian neoplasms.
Aim: To evaluate the clinical significance of methylation levels of 13 lncRNA genes (GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, PLUT/PDX1-AS1, SNHG1, SNHG6, SNHG12, SNHG17, TINCR, TP53TG1, TUG1) associated with various types of ovarian cancer metastasis, including lymphogenous, peritoneal, omental, and distant metastases.
Methods: The methylation levels of lncRNA genes GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, PLUT/PDX1-AS1, SNHG1, SNHG6, SNHG12, SNHG17, TINCR, TP53TG1, TUG1 were analyzed by quantitative real-time methylation-specific polymerase chain reaction. We tested 122 duplicate samples of ovarian neoplasms, including 104 malignancies and 18 borderline tumors, as well as 45 peritoneal macro metastases, collected in the N.N. Blokhin National Medical Research Center of Oncology in 2020 to 2023. The study included 21 samples of primary tumor from patients with lymphogenous metastases, 45 samples from patients with peritoneal dissemination, 61 from those with omental metastases, 49 from patients with ascites, and 9 with distant metastases.
Results: The tumor samples from the patients with lymphatic nodes metastases showed a significant increase in the methylation level of two lncRNA genes: SNHG6 (p = 0.044) and SNHG12 (p = 0.006). Peritoneal dissemination was associated with hypermethylation of four lncRNA genes: GAS5, HOTAIR, LINC00472 (p < 0.05), and most significantly TINCR (p = 0.001). GAS5, HOTAIR, LINC00886 (p < 0.05) and most significantly LINC00472 (p < 0.001) hypermethylation was typical for omental metastasis, and that of LINC00472 and LINC00886, with ascites (p < 0.05). Peritoneal macroscopic metastases demonstrated increased methylation of MAFG-DT (p < 0.001) and TP53TG1 (p < 0.001) and desmethylation of LINC00886 (p = 0.003) and SNHG12 (p = 0.002), compared to their primary tumors.
Conclusion: We performed the analysis of clinical significance of 13 hypermethylated lncRNA genes in ovarian cancer and were the first to show that 10 genes (GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, SNHG6, SNHG12, TINCR, TP53TG1, TUG1) were associated with various types of ovarian tumor metastasis. Also, we were able to determine certain panels of lncRNA, which, if demonstrate abnormal methylation, were specific for lymphogenous and peritoneal metastasis of ovarian tumors.
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Serum concentrations of chromogranin A, serotonin and natriuretic peptide are decreased in patients with neuroendocrine tumors with overweight and obesity compared to healthy donors
Abstract
Background: Neuroendocrine tumors (NETs) are a group of neoplasms, in which circulating biomarkers chromogranin A (CgA), serotonin, and N-terminal brain natriuretic pro-peptide (NT-proBNP), used as a marker of carcinoid heart disease, are especially important in the diagnosis and monitoring. A number of pre-analytical factors, including patients' body mass index (BMI), have an impact on NETs biomarker concentrations.
Aim: To perform a comparative analysis of serum concentrations of CgA, serotonin and NT-proBNP in patients with NETs of various locations with normal body weight and with overweight or obesity.
Methods: This cross-sectional study included 94 patients with NETs of various locations and 78 provisionally healthy individuals without cancer and cardiovascular disorders, matched by gender and age to the patients. Serum biochemical markers were measured before a course of chemotherapy/biotherapy or surgery with the use of standardized enzyme-linked immunosorbent assays Chromogranin A NEOELISA (Eurodiagnostica), Serotonin ELISA (IBL), and the electrochemiluminescent assay (Cobas e601 analyzer, Roche). All NETs patients were divided into 2 subgroups: 46 patients with normal bodyweight (BMI 18 to 24.9) and 48 patients with overweight/obesity (BMI ≥ 25).
Results: The median BMI in the NET patients did not vary depending on their tumor extension, functional activity and malignancy grade. The median concentrations of CgA (150 ng/ml), serotonin (188 ng/ml) and NT-proBNP (117 pg/ml) in the NET patients with obesity and overweight were significantly lower (p = 0.008, p = 0.005 and p = 0.012, respectively) than in the NET patients with normal body weight (769 ng/ml, 704 ng/ml and 197 pg/ml, respectively). In the control group, significantly (p = 0.0001) lower levels in the subgroup with BMI ≥ 25 were obtained only for serotonin. The ROC analysis showed a decrease of the diagnostic efficiency of NET biomarkers in obesity: in the normal BMI group AUC (CgA) was 0.87 and AUC (serotonin) 0.78, whereas in those with BMI ≥ 25, the AUC (CgA) and AUC (serotonin) were 0.81 and 0.62, respectively.
Conclusion: Obesity may make it difficult to identify biochemical markers of NETs – CgA, serotonin and NT-proBNP.
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The LGALS1 gene polymorphism is not associated with galectin-1 levels in tumor tissue and blood of colon cancer patients
Abstract
Background: Galectin-1 plays an important role in the pathogenesis of colorectal cancer (CRC). The blood and tumoral levels of galectin-1 could be dependent on the polymorphism of the promotor region of LGALS1 gene.
Aim: To analyze an association between galectin-1 levels in tumor tissue and plasma and the genotype of the rs4820293 and rs4820294 polymorphisms of the LGALS1 gene in CRC patients.
Materials and methods: The study included a total of 70 inpatients with pathologically verified CRC (International Classification of Diseases 10th Revision codes C18-C20, 39 men and 31 women, mean age 65.4 ± 5.7 years), who were receiving treatment in the Tomsk Regional Oncology Center and Cancer Research Institute of the Tomsk National Research Medical Center from 2020 to 2022. The control group consisted of 70 healthy volunteers (34 men and 36 women, mean age 62.3 ± 7.2 years). Venous blood samples were taken from all study participants and tumor tissue samples were obtained from the CRC patients. Galectin-1 expression in the tumor tissue was assessed by immunohistochemistry and plasma galectin-1 levels by enzyme-linked immunosorbent assay. The LGALS1 gene polymorphisms rs4820293 and rs4820294 were identified by restriction fragment length polymorphism analysis.
Results: The distributions of genotype and allele frequencies of polymorphic variants rs4820293 and rs4820294 of the LGALS1 gene in the CRC patients and in the healthy donors were comparable (p > 0.05). Calculation of odds ratios did not confirm any association between LGALS1 gene polymorphisms and CRC. However, the rs4820294 polymorphism had a strong association with regional metastasis and tumor differentiation grade (Cramer's V above 0.4, p < 0.001). The plasma galectin-1 levels in the CRC patients with the AA genotype of the rs4820294 polymorphism were higher than in the healthy carriers (17.42 versus 12.92 ng/ml, p = 0.040). However, there were no significant differences in the content of galectin-1+ cells in the tumor and galectin-1 in plasma of the CRC patients depending on the genotype of the LGALS1 gene polymorphisms (p > 0.05).
Conclusion: The LGALS1 gene polymorphism is not associated with CRC, but in the carriers of the rs4820294 variant is related to clinical and morphological parameters of the tumor process. The intratumoral expression and blood levels of galectin-1 in CRC patients are not dependent on the genotype of rs4820293 and rs4820294 polymorphisms of the LGALS1 gene.
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REVIEW ARTICLE
Mental disorders of the depressive and anxiety spectrum in neuroendocrine tumors: prevalence, pathogenesis, clinical particulars
Abstract
Neuroendocrine tumors (NETs) are a heterogeneous group of slowly progressing tumors from neuroendocrine cells able to secrete biologically active substances. The most striking clinical manifestation of functioning NETs is the carcinoid syndrome, which is caused by serotonin overproduction and is most often associated with hot flashes, diarrhea, and abdominal pain. The prevalence of depressive and anxiety spectrum disorders in NETs patients can amount to 30–50%. The development of depressive and anxiety spectrum disorders in NETs can occur both through a nosogenic mechanism facilitated by a long pre-diagnostic stage, the presence of a serious disease with severe somatic symptoms and resulting significant decrease in quality of life. The mental disorders may be a result of somatogenic factors, which include an imbalance of significant mediators in the central nervous system (in particular, serotonin), as well as chronic inflammation and abnormalities of the body's immune defense. In the treatment of patients with NETs and verified anxiety and depressive disorders, special attention is paid to the safety of prescribing the first-line therapy, the selective serotonin reuptake inhibitors. The side effects of these agents are related to the activity of serotonin receptor agonists in the gut, which results in diarrhea and increased gastrointestinal motility, the symptoms similar to the carcinoid syndrome manifestations. The accumulated evidence allows concluding on the relative safety of serotoninergic antidepressants in NETs patients, but does not exclude some groups of patients with an increased risk of complications related to an additional effect on serotonin metabolism. Current knowledge on the prevalence, pathogenesis and treatment of depressive and anxiety spectrum disorders, comorbid to NETs, are limited and contradictory. Therefore, further studies with a larger patient samples are required to identify key factors of the pathophysiology and manifestation of mental disorders in NETs patients, which would generally facilitate the optimization of their treatment.
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