Autism Spectrum Disorder Pathogenesis-A Cross-Sectional Literature Review Emphasizing Molecular Aspects.

The etiology of autism spectrum disorder (ASD) has not yet been completely elucidated. Through time, multiple attempts have been made to uncover the causes of ASD. Different theories have been proposed, such as being caused by alterations in the gut-brain axis with an emphasis on gut dysbiosis, post-vaccine complications, and genetic or even autoimmune causes. In this review, we present data covering the main streams that focus on ASD etiology. Data collection occurred in many countries covering ethnically diverse subjects. Moreover, we aimed to show how the progress in genetic techniques influences the explanation of medical White Papers in the ASD area. There is no single evidence-based pathway that results in symptoms of ASD. Patient management has constantly only been symptomatic, and there is no ASD screening apart from symptom-based diagnosis and parent-mediated interventions. Multigene sequencing or epigenetic alterations hold promise in solving the disjointed molecular puzzle. Further research is needed, especially in the field of biogenetics and metabolomic aspects, because young children constitute the patient group most affected by ASD. In summary, to date, molecular research has confirmed multigene dysfunction as the causative factor of ASD, the multigene model with metabolomic influence would explain the heterogeneity in ASD, and it is proposed that ion channel dysfunction could play a core role in ASD pathogenesis.

Could veterinarians be immune to contracting SARS-CoV-2?

Introduction: The aim of the study was to assess the epidemic situation among veterinarians of the Swietokrzyskie Voivodeship, Poland, in relation to the control group. Material and methods: The research was divided into 3 stages. Stage I involved the selection of subjects. In stage II, flow cytometry for immunophenotyping was performed and the percentage of the sub-population of CD4 cells and CD8 cells was assessed. Stage III involved collection of nasopharyngeal swab samples in order to determine the canine coronavirus CR-CoV mRNA with the rT-PCR method. Results: The percentage of the CD4 and CD8 lymphocyte subpopulation in relation to the total lymphocyte population in veterinarians did not differ statistically from the percentage in the control group. The CD4/CD8 ratio in the group of veterinarians was on average 1.93, and 2.04 in the control group. There was no statistically significant difference between the groups, p = 0.591. Canine CR-CoV mRNA was not detected in any of the veterinarians or in the control group. Conclusions: None of the veterinarians had a significant increase in T lymphocytes, which could be an effective defense against SARS-CoV-2.

A five-year follow up study of stage I-IV of rectal cancer with an emphasis on epidermal growth factor over-expression.

The key pro-proliferative pathway, based on EGFR-KRAS/BRAF-myc, is seen as the main goal of personalized therapy in rectal cancer. The objective of the study is to assess the EGFR immunoreactivity in rectal cancer and to estimate its relationship with the clinical outcome, especially as a predictor of poor outcomes. Patients: applying exclusion criteria, 102 patients with stage I-IV rectal cancer, who had undergone scheduled surgery during the period 2005-2011, were included in the study. There was a follow-up study with a span of 5 years from the date of the surgery. Immunohistochemistry using EGFR (EGFR Ab10, Clone111.6) was performed to detect an overexpression of the targeted receptor. Digital analysis of positive reactions of membranes was performed utilizing VisiopharmTM. The degree of EGFR intensity (log OR 0.854, OR 2.35, 95% Cl: 1.14-4.85, p = 0.021) is a significant factor in the prognosis of death within 2 years of surgery. The OS curve showed a significant decrease after 40 months from the date of surgery in the cases where EGFR had a high expression. The ROC curve for the cancer stage, according to the UICC classification and EGFR expression, in order to predict a 2-year RFS, reached a high specificity value (ROC = 0.81, p = 0.0408). Immunohistochemical EGFR expression is inexpensive, specific and broadly available.

P21 is not a prognostic marker for rectal cancer - five-year follow up study of rectal cancer in stages I-IV.

The p21 participates in the regulation of DNA repair and replication, and modulation of apoptosis as well. After DNA damage, the p53-dependent induction of p21 results in cell cycle arrest or could trigger cell apoptosis. The objective of the study was the assessment of p21 immunoreactivity in rectal cancer and the estimation of relationships with clinical outcome especially as predictor of poor outcome. While applying the ruling in and out criteria, 102 patients were incorporated to the study, with stage I-IV rectal cancer who had undergone surgery in a planned mode during 2005-2011. The follow-up covered 5 years period from surgery date. Conventional immunohistochemistry were performed using antibody against p21 (p21WAF1 (Clone H252) to detect overexpression targeted receptor. The analysis showed no statistically significant differences in the survival curves of patients in groups with immunoreactivity of p21 protein at 0; 1; 2; 3 (p = 0.6453 in the log-rank test), also is not a significant risk factor for death (HR = 0.915, p = 0.7842) and for tumor dissemination (HR = 0.94, p = 0.9426). Our study leads to the conclusion that the probability of survival does not depend on p21 expression and do not authorize the importance of p21 immunoreactivity in the detection and monitoring of rectal cancer treatment.

Gastric gastrointestinal stromal tumor with incomplete duplication cyst - a case with possibility of neoplasia in fetal-period malformed tissues.

The coincidence of GIST and other gastric malignancies are documented well but arising GIST from congenital anomalies is still rarity in literature. To date, only a few papers have been concerned on the possibility of arising neoplasms from duplication cyst of gastrointestinal tract. There, are dominating usual cancers, neuroendocrine cancers or lymphomas but GIST has been noted only once. Here, we report a case of 73 years old female-patient with typical gastric stromal tumor comprised centrally locked an incomplete duplication cyst.

Vulvar angiomyofibroblastoma--a case report of rare entity mimicking Bartholin cyst.

Vulvar angiomyofibroblastoma is rare tumor of obscure histological origin. Here a case of 49-year old woman is described with this intriguing benign vulvar entity. The tumor developed at left vulvar labia and clinically imitated Bartholin cyst with clinical complaints of regional discomfort without pain. A macroscopic evaluation revealed well separated, encapsulated tumor of 3,5 cm in diameter. On cut surface the tumor was whitish, flesh, solid with myxoid appearance without any apparent cysts formation. There were alternating hypo- and hypercellular in the neoplasm. Microscopically the tumor comprised proliferation of small thin walled vessels that were surrounded with cuffs and islands of epithelioid, spindle and plasmacytoid cells with occasional vacuolization. Some aggregations of cells were quite dense and in such fields, vessels were compressed and ecstatic enough to mimic a bit haemangiopericytoma pattern. A production of myxoid intercellular matrix was seen in loose, hypocellular areas and was confirmed by positive pas-alcian blue stain that demonstrated prominent myxoid stroma and intracytoplasmatic globules of acid glicoproteins. The immunoprofile was remarkable enough to show strong expression of vimentin and desmin, while there was a lack of pan-keratin (CKAE1/3) and smooth muscle actin (SMA) immunoreactivities. Such an immunofentype is regarded to share some of myofibrolastic origin despite SMA negativity. Tumor cells seemed to sprout from perivascular regions giving an impression of accumulations strictly associated with neighbouring vascular branches. This configuration of cells is very often viewed as pericyte-like proliferation. Thus, our case of angiomyofibroblastoma is an example of tumor that probably derives from perivascular stem cells that acquire some of myoid features.

NOS2 Polymorphism in Aspect of Left and Right-Sided Colorectal Cancer.

Background: The NOS2 gene polymorphism rs2297518 is associated with an increased level of NO, which could contribute to colorectal cancer (CRC) development. We hypothesized that the potential influence of the NOS2 gene polymorphism on cancer development may vary between right-sided and left-sided colon cancers, and rectal cancers. The aim of this study was to determine the rs2297518 polymorphism influence on colorectal cancer development with regard to tumor localization. Methods: This case-control study included 199 patients with CRC and 120 controls. The qPCR endpoint genotyping was conducted using the TaqMan® genotyping assay. Results: This study revealed significant differences in tumor characteristic and in the minor alelle A frequency in the NOS2 genotype between colorectal cancers with different localizations. The mucinous adenocarcinoma was diagnosed significantly more often in right-sided cancers than in left-sided (30.6% vs. 10.9%, p = 0.009) and rectal cancers (30.6% vs. 7.1%, p = 0.0003). The minor allele A of the NOS2 genotype was observed more frequently in right-sided cancers than in left-sided cancers (44.9% vs. 23.1%, p = 0.0137) and more frequently in rectal cancers than in left-sided cancers (40.0% vs. 23.1%, p = 0.0285). Conclusions: In conclusion, the results support the hypothesis that the SNP rs2297518 of the NOS2 gene influences colorectal cancer development with regard to tumor localization.

Comparison of primarily diet-modifiable intestinal factors, connexin 43 and E-cadherin with TP53 and TGFB1 in colorectal cancer.

BACKGROUND/AIMS: Primarily, a diet (particularly dietary lipids and vitamins) can reversibly modify intestinal expressions of a few factors like connexin 43 (Cx43), E-cadherin (Cdh1), TP53 and TGFB1 with a special impact on immunity and mutagenesis. Malignant phenotype constitutes a diet-resistant signal streaming with engagement of these molecules which are generated in autonomous ways in colorectal cancer. METHODOLOGY: We aimed to compare adhesion proteins: (Cx43) and Cdh1 with TP53 and TGFB1 in colorectal adenocarcinomas. GJA1P1 and Cdh1 with TP53 and TGFB1 were detected with immunohistochemistry in the study of 106 colorectal adenocarcinomas. RESULTS: There was aberrant cytoplasmic expression instead of membranous one of Cx43 and Cdh1 reflecting constitutive destruction of intercellular ties while TP53 showed nuclear expression and TGFB1 accumulated in the cytoplasm. TP53 did not correlate with Cx43 (r=0.083, p=0.397) but correlated with Cdh1 (r=0.199, p=0.041). Cdh1 associated with TGFB1 reaching almost statistical significance (r=0.188, p=0.054), while TGFB1 correlated with Cx43 (r=0.359, p=0.001). CONCLUSIONS: The consequent and constant impairment of cancer intercellular communication seems to engage correlated with each other expressions of Cx43 and TGFB1 in colorectal cancer cells.

Sexual Behavior and the Awareness Level of Common Risk Factors for the Development of Cervical, Anogenital and Oropharyngeal Cancer among Women Subjected to HR HPV DNA-Testing.

The human papillomavirus (HPV) is a significant health problem that increases the risk of developing precancerous lesions, cancer of the anogenital area, as well as mouth and throat cancers. The aim of the study was to analyze the awareness level of common risk factors and the preferred sexual behavior of women aged 18-68, who underwent a molecular evaluation of common HR-HPV from material collected from the cervix. The study was conducted at the Jan Kochanowski University in Kielce, Collegium Medicum, in the period from December 2019 to August 2020 on a group of 201 women. A diagnostic survey and the HR-HPV molecular test were used in the research. All of the obtained samples were subjected to identification of and testing for the presence of HR-HPV by the Cobas 4800 platform (Roche Diagnostic©, Basel, Switzerland). We presented the statistically significant relationships between the age of the respondents and the awareness of the impact. The women aged over 43 years old presented the widest spectrum of information concerning HPV-related lesions. Conclusion: Our study highlights the necessity of educating women about the prevention of sexually transmitted infections.

Endometrial Cancer in Aspect of Forkhead Box Protein Contribution.

(1) Background: The present study aimed to investigate the influence of forkhead box (FOX) on endometrial cancer (EC) progression. For a better understanding, the driving mechanisms are vital to identifying correlations between genes and their regulators. (2) Methods: The study enrolled one hundred and three white female patients with confirmed EC. For the analysis, we used next-generation sequencing with the Hot Spot Cancer Panel provided by Illumina Inc., San Diego, CA, USA, and an immunohistochemical analysis of FOXA1, FOXP1, and estrogen receptors. (3) Results: FOXA1 silencing led to a worse outcome based on the correlation with FOXA1 (test log-rank p = 0.04220 and HR 2.66, p = 0.033). Moreover, FOX proteins were closely correlated with TP53 and KRAS mutation. (4) Conclusions: Our study confirmed previous reports about FOX box protein in the regulation of tumor growth. A remarkable observation about the unclear crosstalk with crucial genes, as TP53 and KRAS need deeper investigation.

FGFR-2 and Epithelial-Mesenchymal Transition in Endometrial Cancer.

BACKGROUND: At present, EC staging is based on the WHO conservative criteria, which only consider the percentage of gland formation. The molecular subgrouping of EC recently proposed by the Cancer Genome Atlas (TCGA) represents a milestone in precise molecular-based patient triage. The present study aimed to investigate the influence of FGFR-2 on the epithelial-mesenchymal transition (EMT) and whether it can lead to endometrial cancer dedifferentiation. METHODS: One hundred and three White female patients with confirmed EC were enrolled in our research. For the analysis, we performed next-generation sequencing and immunohistochemical analyses of E-cadherin, ß-catenin, and vimentin. RESULTS: Tumor grade progression was closely correlated with LVI (p = 0.0338), expression of vimentin (p = 0.000), tumor budding (p = 0.000), and lack of E-cadherin (p = 0.0028). Similar observations were noted with regard to TNM/FIGO stage progression. In terms of FGFR-2 mutation, we found the following correlation p-values: LVI (p = 0.069), expression of vimentin (p = 0.000), tumor budding (p = 0.000), and lack of E-cadherin (p = 0.000), RFS (p = 0.032), ECSS (p = 0.047). CONCLUSIONS: FGFR-2 is the important factor influencing on EMT.

The Roles of TP53 and FGFR2 in Progress Made Treating Endometrial Cancer.

The morbidity and mortality caused by endometrial cancer (EC) is still rising worldwide. In recent years, a new system of tumor stratification has been proposed based on POLE-mutational status, TP53, and microsatellite stability status. The aim of the study was to analyze a vast panel on the genes potentially involved in the genesis of endometrial cancer in the Polish population. One hundred and three white female patients with confirmed endometrial cancer were enrolled on the study. We performed sequencing using the Hot Spot Illumina panel and microsatellite stability with immunohistochemistry. We confirmed a key role of the TP53 mutation in progress to high-grade EC and parallelly some role of FGFR2 mutation. Moreover, our data present a vast landscape of mutations in EC and their polymorphism. We reported the meaning of FGFR2 mutation and TP53 (high copy number) in high-grade ECs. Our observation in MSI contribution is comparable with other studies. Finally, we see a strong need for the implementation of the TCGA classification.

Five-year follow-up study of stage I-IV rectal cancer including EGFR immunoexpression and p21 immunoactivity.

INTRODUCTION: Both environmental and genetic factors increase the likelihood of developing rectal cancer. AIM: To assess the EGFR and p21 immunoreactivity in rectal cancer and to assess its relationship with the clinical outcome. MATERIAL AND METHODS: Applying exclusion criteria, 102 patients with stage I-IV rectal cancer, who had undergone scheduled surgery during the period 2005-2011, were included in the study. There was a follow-up study with a span of 5 years from the date of the surgery. Immunohistochemistry using epidermal growth factor receptor (EGFR Ab10, Clone111.6) and antibodies against p21 (p21WAF1 (Clone H252)) was performed to detect overexpression of the targeted receptor. Digital analysis of positive reactions of membranes and nuclei was performed utilizing Visiopharm. RESULTS: The degree of EGFR intensity (log OR = 0.854, OR = 2.35, 95% CI: 1.14-4.85, p = 0.021) is a significant factor in the prognosis of death within 2 years after surgery. The OS curve showed a significant decrease after 40 months from the date of surgery in the cases where EGFR had high expression. The ROC curve for cancer stage, according to the UICC classification and EGFR expression, in order to predict 2-year RFS, reached a high specificity value (ROC = 0.81, p = 0.0408). The analysis showed no statistically significant differences in the survival curves of patients in groups with immunoreactivity of p21 protein at 0, 1, 2, 3 (p = 0.6453 in the log-rank test). Also, it is not a significant risk factor for death (HR = 0.915, p = 0.7842) or for tumor dissemination (HR = 0.94, p = 0.9426). CONCLUSIONS: The determination of EGFR immunoreactivity is important in the monitoring and treatment of patients with rectal cancer, as opposed to p21.

GERD-A Burning Problem after Sleeve Gastrectomy?

BACKGROUND: Surgical treatment is the most effective method of treatment for obesity; and laparoscopic sleeve gastrectomy (LSG) is the most frequently performed bariatric surgery. OBJECTIVE: The aim of the study was evaluation of the frequency of occurrence and the degree of progression of changes characteristic of GERD in patients who had undergone LSG in clinical; endoscopic; and microscopic images in the obtained bioptats; and an attempt to correlate the results obtained with the effectiveness of bariatric treatment. MATERIALS AND METHOD: The anonymized clinical data concerning 214 patients who had undergone LSG were collected from the database. Invitations for check-up examinations were distributed, to which 37 patients responded. Two patients were excluded from the study. In 35 patients after LSG check-up clinical examination, bariatric endoscopy (BE) and bariatric microscopy (BM) were performed on bioptats collected from the site of the gastro-esophageal junction; and 3 specimens collected at 2 cm intervals from the Z-line. The study was performed according to the standard protocol. RESULTS: In the analyzed group, clinical symptoms of GERD occurred in 12 patients (34.5%), including 10 females and 2 males. The symptom reported by all patients was retrosternal pain/burning (heartburn). In BE, foci of ectopic mucosa in the epicardial part of the esophagus were found in 16 patients (14 F and 2 M). No correlation was observed between the analyzed parameters and the occurrence of the above-mentioned changes. In BM, only in three patients were the changes described as normal esophageal mucosa; while in another three, as foci of intestinal metaplasia, Barrett's esophagus. In this group no foci of dysplasia were found. In eight patients, the changes were described as inflammatory. In ten patients from this group, microscopic changes occurred without clinical symptoms of the disease. CONCLUSIONS: GERD is an important clinical problem in patients after LSG; therefore; the problem of occurrence or exacerbation of symptoms of the disease should be discussed with the patient during qualification for bariatric surgery. The bariatric effectiveness of LSG does not correlate with the occurrence of the symptoms of GERD after the procedure. However; the lack of clinical symptoms of the disease does not mean the lack of its occurrence. Therefore; the endoscopic check-up after LSG should be routinely performed. During the qualification for LSG screening, histopathologic examinations of the esophagus may be useful for the assessment of the microscopic symptoms of GERD in oligosymptomatic patients; and exclusion of rare pathologies of the esophagus (e.g., eosinophilic esophagitis), which may complicate post-operative course.

A New Aberration in the VPS33B Gene Leads to Full-Symptom ARCS1.

BACKGROUND ARCS1 is an acronym for arthrogryposis, renal dysfunction, and cholestasis. It is a congenital malfunction with autosomal recessive inheritance, and, unfortunately, its prognosis is still poor. It is believed that VPS33B is altered in 75% of cases and that the VIPAR gene is altered in approximately 25% of them. CASE REPORT An affected child was born from the first pregnancy of 26-year-old mother and a 30-year-old father with no previous medical history and no genetic conditions. The first clinical symptoms were observed at the end of the child's second week of life. The mother reported the child has decreasing body weight and loss of appetite. After admission to the ward, the child was apathetic and sleepy. Symptoms of conjunctivitis, pale and dry skin, and mild face and mild body dysmorphia were observed. CONCLUSIONS Laboratory tests revealed proteinuria of up to 1.36 g/l and glycosuria of up to 28 mmol/l, as well as fluctuating metabolic acidosis. The bilirubin level reached 6.62 mg/dl, along with alkaline phosphatase at 470 U/l. Moreover, hypothyroidism with TSH at 16.71 uU/ml was observed. Because of the co-occurrence of cholestasis and renal dysfunction, molecular testing was done. The 17th exon of VPS33B was sequenced by Sanger DNA sequencing method. To the best of our knowledge, this is the first report of homozygotic mutation c.1235_1236delinsG (p.Pro412ArgfsTer7) in the VPS33B gene. The risk of transfer of the mutation to future descendants was calculated as 25%. Due to the wide landscape of molecular alternation in the 17th exon of the VPS33B gene, we propose using Sanger whole-exon sequencing as a first-choice diagnostic test.

HPV genotyping and p16/Ki-67 test significantly improve detection rate of high-grade cervical squamous intraepithelial lesion.

INTRODUCTION: Liquid-based cytology allows to apply modern and specific analyses of hrHPV genotyping in p16/Ki-67 test. All of these together could raise accuracy ratio for high-grade squamous intraepithelial lesion above 90%. The purpose of this study was to evaluate the diagnostic accuracy of LBC, hrHPV testing, and p16/Ki-67 testing in diagnosis of high-grade cervical intraepithelial lesions. MATERIAL AND METHODS: The study consisted of 176 women, out of which 50 presented with HSIL (CIN2) SCC (cervical intraepithelial lesion grade 2 squamous cell carcinoma). 126 women with a negative Pap test were pooled into the second group of the study. All patients were resampled for LBC, HPV genotyping, and for the p16/Ki-67 test. The research was carried out between May and December 2017, and second sampling were taken from 1 to 4 months. RESULTS: We reported a strong correlation between positive Pap test and hrHPV (p < 0.05) that met accuracy close to 90%. We noted correlations between a positive p16/Ki-67 with a positive Pap test: p < 0.001; 66% sensitivity (95% CI: 51.2-78.8%), 87.8% specificity (95% CI: 75.2-95.4%), 76.8% accuracy (95% CI: 67.2-84.7%), and OR 13.9 (95% CI: 4.9-39.2), especially HSIL and HPV16: p < 0.001; sensitivity (95% CI) 64.0, specificity (95% CI) 98.4, accuracy (95% CI) 88.6, OR (95% CI) 109.3. CONCLUSIONS: The results of our study indicate hrHPV genotyping as a good biomarker for the triage of patients with an abnormal cytological report. In our opinion, the hrHPV test reaches the highest level of sensitivity, specificity, and accuracy, and should be considered as crucial diagnostic test in cervical screening.

The Influence of Red Meat on Colorectal Cancer Occurrence Is Dependent on the Genetic Polymorphisms of S-Glutathione Transferase Genes.

BACKGROUND: It is postulated that both individual genotype and environmental factors such as diet may modify the risk of developing colorectal cancer (CRC). The influences of GST gene polymorphism and red meat intake on CRC occurrence in the Polish population were analyzed in this study. METHODS: Genotyping was performed with the qPCR method. RESULTS: A high frequency of meat consumption was associated with an over 2-fold increase in the risk of colorectal cancer odds ratio (OR) adjusted for sex and age = 2.4, 95% confidence interval (CI); 1.3-4.4). However, after analyzing the genetic profiles, in the absence of polymorphisms of all three analyzed genes, there was no association between a high frequency of meat consumption and the occurrence of CRC. In the case of GSTM1 gene polymorphism, the high frequency of meat consumption increased the risk of CRC by almost more than 4 times (OR adjusted for sex and age = 3.8, 95% CI: 1.6-9.1). For GSTP1 gene polymorphism, a 3-fold increase in CRC risk was observed with a high frequency of meat consumption (OR adjusted for sex and age = 3.4, 95% CI: 1.4-8.1). In the case of GSTT1 gene polymorphism, the increase in risk of CRC was not statistically significant (OR adjusted for sex and age = 1.9, 95% CI: 0.4-8.5). CONCLUSIONS: The frequency of red meat intake in non-smokers increases the risk of colon cancer in the case of GST gene polymorphisms.

GSTM1, GSTT1, and GSTP1 polymorphisms and colorectal cancer risk in Polish nonsmokers.

Glutathione S-transferase (GST) enzymes are responsible for cellular detoxification of many carcinogens and are important anticancer elements. This study assessed potential relationships between GSTM1, GSTT1, and GSTP1 polymorphisms and colorectal cancer (CRC) risk in Polish nonsmokers. We also analyzed the influence of GST gene polymorphisms on CRC clinical and histopathological features. Our study included 197 CRC patients and 104 healthy controls. GSTM1, GSTT1, and GSTP1 polymorphisms were evaluated using qPCR. Polymorphism frequencies observed in our control group corresponded to those in other European populations. The GSTM1 null and GSTT1 null genotypes were observed with similar frequencies in both CRC patients and controls (GSTM1 null: 46.7% vs. 45.2%; GSTT1 null: 15.7% vs. 20.2%). GSTP1 Ile/Ile, Ile/Val, and Val/Val genotype frequencies were respectively 42.1%, 48.2%, and 9.6% in patients and 48.1%, 42.3%, and 9.6% in controls. GSTT1 polymorphism correlated with higher tumor grade in CRC patients, and the GSTM1 null/null genotype was associated with more frequent metastasis to lymph nodes (pN classification). Our results suggest that GST gene polymorphisms may influence CRC tumor grade and stage.

Tumor Digital Masking Allows Precise Patient Triaging: A Study Based on Ki-67 Scoring in Gastrointestinal Stromal Tumors.

BACKGROUND: Technological advances constantly provide cutting-edge tools that enhance the progress of diagnostic capabilities. Gastrointestinal stromal tumors belong to a family of mesenchymal tumors where patient triaging is still based on traditional criteria such as mitotic count, tumor size, and tumor location. Limitations of the human eye and randomness in choice of area for mitotic figure counting compel us to seek more objective solutions such as digital image analysis. Presently, the labelling of proliferative activity is becoming a routine task amidst many cancers. The purpose of the present study was to compare the traditional method of prediction based on mitotic ratio with digital image analysis of cell cycle-dependent proteins. METHODS: Fifty-seven eligible cases were enrolled. Furthermore, a digital analysis of previously performed whole tissue section immunohistochemical assays was executed. Digital labelling covered both hotspots and not-hotspots equally. RESULTS: We noted a significant diversity of proliferative activities, and consequently, the results pointed to 6.5% of Ki-67, counted in hotspots, as the optimal cut-off for low-high-grade GIST. ROC analysis (AUC = 0.913; 95% CI: 0.828-0.997, p < 0.00001) and odds ratio (OR = 40.0, 95% CI: 6.7-237.3, p < 0.0001) pointed to Ki-67 16% as the cut-off for very high-grade (groups 5-6) cases. With help of a tumor digital map, we revealed possible errors resulting from a wrong choice of field for analysis. We confirmed that Ki-67 scores are in line with the level of intracellular metabolism that could be used as the additional biomarker. CONCLUSIONS: Tumor digital masking is very promising solution for repeatable and objective labelling. Software adjustments of nuclear shape, outlines, size, etc. are helpful to omit other Ki-67-positive cells especially small lymphocytes. Our results pointed to Ki-67 as a good biomarker in GIST, but concurrently, we noted significant differences in used digital approaches which could lead to unequivocal results.