NRAS Genes Research Articles

A Novel NRAS Variant Near the Splice Junction in Moroccan Childhood Acute Lymphoblastic Leukemia: A Molecular Dynamics Study.

The RAS genes are importantly implicated in oncogenesis and are frequently mutated in childhood acute lymphoblastic leukemia. This study is the first to our knowledge, to determine the mutational status of NRAS and KRAS genes in Moroccan pediatric acute lymphoblastic leukemia (ALL). Polymerase chain reaction and Sanger sequencing were performed for 45 ALL samples to explore the coding exons. The functional effect of the mutation was evaluated using in silico prediction tools and molecular modeling. We identified a novel variant c.290 G > C p.Arg97Thr within NRAS gene in a patient with T-ALL, which is a rare missense point mutation affecting the last base of exon 3. Analyses revealed that p.Arg97Thr impairs the adjacent splice site efficiency. Moreover, it leads to structural modifications at local and global levels of the protein through the loss of hydrogen bonds. Additionally, the molecular dynamics (MD) simulation showed that it slightly increases the stability of NRAS protein by locally decreasing the flexibility of the mutated region. No variant was detected within KRAS gene. R97 at NRAS gene is an overlapping splice site residue. Our findings suggest that the NRAS p.Arg97Thr variant may disrupt the splicing machinery and functions of the protein, thus playing a vital role in leukemogenesis. In addition, the highly druggable pocket may possibly be studied for its therapeutic implications.

The genomic landscape of papillary thyroid carcinoma on next-generation sequencing in patients undergoing total thyroidectomy.

Thyroidectomy is increasingly performed for suspected malignancy. This cohort study aimed to identify genetic markers associated with malignancy and determine the molecular landscape of papillary thyroid carcinoma (PTC) through next-generation sequencing (NGS) in patients undergoing total thyroidectomy. Among 116 surgical candidates, 103 patients (age=42.9±13.7years; Male: Female=14:89) with benign or malignant thyroid nodules were eligible. Live thyroid tissue samples harvested intraoperatively with adequate DNA and RNA yield were subjected to NGS on the Illumina NovaSeq 6000 platform for genomic and transcriptomic analysis, respectively. Histopathology comprised 20 malignant (19.4%) and 83 benign (80.6%) cases, including 16 PTC (15.5%) cases. On NGS, single nucleotidepolymorphisms (SNPs) in NTRK1 at NC_000001.11:156879016 on chromosome 1 and ALK at NC_000002.12:29717663 on chromosome 2 were frequent in malignant lesions (p<0.05). A SNP in ALK at NC_000002.12:29193706 was consistently a homozygous alternate alleleacross the cohort. DNA-sequencing of PTC lesions identified recurrentsomatic mutations in BRAF (100%), ALK (56.3%), RET (18.8%), PIK3CA (12.5%), NTRK1 (12.5%), NTRK2 (87.5%), NTRK3(12.5%), NRAS(6.3%), and PTCH1 (31.3%) genes. RNA sequencing revealednovelfusiongenes, including MKRN1-BRAF, RN7SL1-CDH1, IRF2BPL-MED12, MED12-IRF2BPL, CPM-MDM2, and AC005895.3-PDGFRB. Inreceiveroperative characteristics analysis, the AUCs ofALKmutationpredictingrecurrence and metastases were 0.818 and 0.783. This Indian study identified novel somatic mutations and fusion genes in PTC, revealing a distinct genomic landscape with implications in precision diagnostics and personalized therapies. NGS with intraoperative live sampling shows promise in prognostication and therapeutic optimization of advanced/metastatic PTC cases. CTRI/2020/09/027607dt 04/09/2020; REF/2020/08/036119.

The discovery and simulation analysis of a novel mutation c.40 G < T (V14F) in the NRAS gene in patients with colorectal cancer in Saudi Arabia

BackgroundColorectal cancer (CRC) is the most diagnosed cancer in men and the second most common cancer in women and remains associated with high morbidity and mortality in Saudi Arabia. The current understanding of genetic heterogeneity of colorectal cancer biology encourages the identification of the genetic causes of CRC in the Saudi population. MethodsIn this study, we ascertained 89 CRC patients’ tumor samples from Saudi Arabia and investigated the molecular alterations of the NRAS proto-oncogene, GTPase (NRAS) gene by sequencing the collected CRC tumor tissue samples to identify gene mutations. The impact of mutations was analyzed using different bioinformatics tools including Missense3D algorithm of Swiss Model platform, molecular dynamics simulations using YASARA DYNAMICS and Protein Variation Effect Analyzer (PROVEAN) tool. ResultsWe identified a novel mutation c.40 G > T, in one patient in whom valine was replaced by phenylalanine (V14F). Notably, we also identified another mutation in the same codon c.40 G > A where valine is replaced by isoleucine (V14I). Our in-silico analysis revealed that this novel mutation alters the binding affinity of the NRAS gene substantially, and as a result, could have lethal consequences on the downstream signaling genes and pathways including MAPK and PI3K involved in regulating CRC growth and progression. ConclusionsThese findings provide insights into the molecular etiology of CRC in general and particularly in the Saudi population. Thus, these findings in NRAS mutation testing may also guide further treatment modalities, and more personalized therapy may be optimized.

Extracellular vesicles powered cancer immunotherapy: Targeted delivery of adenovirus-based cancer vaccine in humanized melanoma model.

Malignant melanoma, a rapidly spreading form of skin cancer, is becoming more prevalent worldwide. While surgery is successful in treating early-stage melanoma, patients with advanced disease have only a 20% chance of surviving beyond five years. Melanomas with mutations in the NRAS gene are characterized for a more aggressive tumor biology, poorer prognosis and shorter survival. Hence, new therapeutic strategies are needed, especially for this specific group of patients. Novel approaches, such as cancer vaccines, offer promising solutions by stimulating the anti-tumor immune response. Nevertheless, their clinical efficacy is still modest and more effective approaches are required. Herein, we propose the systemic administration of the adenovirus-based cancer vaccine complexed in extracellular vesicles (EVs) with the aim of achieving a targeted therapeutic effect. The vaccine was based on previously tested oncolytic adenovirus Ad5/3-D24-ICOSL-CD40L in combination with melanoma-specific antigens targeting NRAS mutations to enhance the anticancer effect. The antineoplastic properties of the oncolytic vaccine were evaluated in xenograft MUG Mel-2 melanoma BALB/c nude mice. Moreover, to mimic the tumor microenvironment, while investigating at the same time immune cell infiltration and drug penetration, we established a 3D co-culture model based on human NRAS mutated MUG Mel-2 spheroids and PBMCs (HLA matched), which displayed a synergistic effect when treated with the cancer vaccine compared to relative controls. Subsequently, we investigated the systemic delivery of the vaccine in EV formulations in a humanized NSG MUG Mel-2 melanoma mouse model. Our study provides a promising strategy for a tumor-targeted vaccine delivery by EVs, resulting in improved anticancer efficacy and increased infiltration of tumor-infiltrating lymphocytes. This study explores the potential of EVs for the selective delivery of cancer vaccines against malignancies, such as NRAS melanoma. Overall, this research could pave the way for applying autologous EVs as a safe and efficacious tool for targeted cancer therapy.

Cutis Verticis Gyrata Differential Diagnosis: Clinical Case

Background. Cutis verticis gyrata is rare benign scalp disorder characterized by excessive skin and subcutaneous tissue proliferation and hypertrophy. Nowadays, there are three forms of this disease with various developmental mechanisms and associated manifestations. Clinical case description. This article presents the patient admitted with diagnosis and clinical picture of pachydermia, who underwent surgical excision of scalp abnormal tissues. However, the diagnosis of giant congenital melanocytic nevus was established according to the results of clinical examination. Later it was confirmed by histological and immunohistological studies. Moreover, pathogenic variants in the NRAS gene were not revealed. Conclusion. This case demonstrates the complexity of differential diagnosis of pachydermia that leads to careful examination of patients with morphogenetic studies of affected tissues samples.

The prominent pervasive oncogenic role and tissue specific permissiveness of RAS gene mutations

In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness.

Amelanotic Melanoma-Biochemical and Molecular Induction Pathways.

Amelanotic melanoma (AM) is a subtype of hypomelanotic or completely amelanotic melanoma. AM is a rare subtype of melanoma that exhibits a higher recurrence rate and aggressiveness as well as worse surveillance than typical melanoma. AM shows a dysregulation of melanin production, cell cycle control, and apoptosis pathways. Knowing these pathways has an application in medicine due to targeted therapies based on the inhibiting elements of the abovementioned pathways. Therefore, we summarized and discussed AM biochemical and molecular induction pathways and personalized medicine approaches, clinical management, and future directions due to the fact that AM is relatively rare. AM is commonly misdiagnosed. Hence, the role of biomarkers is becoming significant. Nonetheless, there is a shortage of biomarkers specific to AM. BRAF, NRAS, and c-KIT genes are the main targets of therapy. However, the role of BRAF and KIT in AM varied among studies. BRAF inhibitors combined with MAK inhibitors demonstrate better results. Immune checkpoint inhibitors targeting CTLA-4 combined with a programmed death receptor 1 (PD-1) show better outcomes than separately. Fecal microbiota transplantation may overcome resistance to immune checkpoint therapy of AM. Immune-modulatory vaccines against indoleamine 2,3-dioxygenase (IDO) and PD ligand (PD-L1) combined with nivolumab may be efficient in melanoma treatment.

Myeloid Sarcoma of Conjunctiva in a patient with JAK2-positive myeloid neoplasm: two distinct myeloid neoplasms sequentially arising from a common precursor

Abstract Introduction/Objective Myeloid sarcoma (aka granulocytic sarcoma or chloroma), a rare type of myeloid neoplasm is characterized by the presence of myelogenous blastic cells outside of the bone marrow either before, during or after the development of leukemia. Presentation in the orbit is extremely rare, unlike other body parts. We report a case of myeloid sarcoma of conjunctiva in a patient with JAK2-positive myeloid neoplasm. Methods/Case Report An 84-year-old man presented with bilateral conjunctival masses which on biopsy showed diffuse involvement by blasts, immunohistochemically expressing positivity for myeloperoxidase, CD33, CD68, CD34 (partial), CD117 (partial) but were non-reactive to CD15, CD3 and PAX5. Next generation sequencing (NGS) revealed mutations in NRAS, ASXl1 and TET2 genes, consistent with the diagnosis of myeloid sarcoma (MS) of the conjunctiva. While patient’s past medical history included status post radiation and chemotherapy for prostate cancer and colonic adenocarcinoma respectively. it was also significant for a JAK 2 positive myeloproliferative neoplasm (MPN), six-years prior to current presentation. At the time he was treated with hydroxyurea for 5 years but developed neutrophilic leukocytosis and transfusion-dependent anemia. Subsequently a treatment regimen including ruxolitinib and pacritinib was initiated but without clinical and hematological improvements. NGS of the initial JAK2-positive MPN revealed ASXL1, JAK2 and TET2 mutations. Of note are the similarities and variations in the molecular signatures of patient’s initial MPN and current conjunctival MS. While the two neoplasms carried an identical TET2 mutation with highest variant allelic frequency, they are different in other mutations. Results (if a Case Study enter NA) NA Conclusion Our case illustrates an unusual manifestation of myeloid sarcoma localized to the conjunctiva. The identification of clonal relatedness on NGS with distinct mutations, adds complexity to our understanding of tumor origin. It highlights a series of events which may have contributed to this phenomenon, including consequence of therapy, clonal evolution with molecular discordance or origin as a totally independent entity. This report underscores the importance of genetic profiling in guiding prognosis and treatment strategies in such settings.

Prognostic impact of next-generation sequencing on myelodysplastic syndrome: A single-center experience.

Myelodysplastic syndromes (MDS) are clinically heterogeneous disorders characterized by peripheral blood cytopenias, poor differentiation, clonal hematopoiesis, and increased risk of developing acute myeloid leukemia (AML). While somatic mutations do not currently feature in prognostic scoring systems, they may impact the clinical phenotype. In recent years, next-generation sequencing (NGS) has enabled the opportunity to identify an increasing number of genetic abnormalities, including recurrent modifications in the TP53, DNMT3A, NRAS, NPM1, RUNX1, and FLT3 genes. Bone marrow aspirate samples of 56 patients with MDS were investigated for mutations using NGS. We compared the relationship between gene mutation status and laboratory characteristics, such as certain cytopenias, the revised international prognostic scoring system, MDS subtypes, karyotypes, AML development, and overall survival. Twenty-one genes were found to have gene mutations, including ASXL1, TET2, SRSF2, EZH2, CSF3R, NRAS, ETV6, SETBP1, RUNX1, DDX41, U2AF1, JAK2, FLT3ITD, SF3B1, DNAMT3A, PHF6, TP53, CEBPA, CBL, IDH2, and GATA2. At least one point mutation occurred in 64.2% of all patients, including 58.3% of those with normal cytogenetics. Thrombocytopenia (P = .016), anemia (P = .018), decreased overall survival (P = .017), and increased AML transformation (P = .023) have been revealed to be linked to non-SF3B1 mutations. MDS are frequently associated with somatic point mutations. According to early findings, NGS panels are extremely effective instruments that provide an entirely new viewpoint on the disease for particular individuals. Future prognostications will depend more on NGS because those who exhibit normal cytogenetics may additionally have gene mutations.

Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma.

Background: Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating BRAF mutations. Mutations in the BRAF gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline BRAF inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic BRAF V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the NRAS gene. Methods: We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in BRAFV600E/NRASQ61K resistant A375 cells. Results: We utilize an AsCas12a protospacer adjacent motif site created by the NRAS Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective NRAS targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. Conclusion: Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.

Two Decades of Progress in Personalized Medicine of Colorectal Cancer in Serbia-Insights from the Institute for Oncology and Radiology of Serbia.

It is projected that, by 2040, the number of new cases of colorectal cancer (CRC) will increase to 3.2 million, and the number of deaths to 1.6 million, highlighting the need for prevention strategies, early detection and adequate follow-up. In this study, we aimed to provide an overview of the progress in personalized medicine of CRC in Serbia, with results and insights from the Institute for Oncology and Radiology of Serbia (IORS), and to propose guidance for tackling observed challenges in the future. Epidemiological data were derived from official global and national cancer registries and IORS electronic medical records. Germline genetic testing for Lynch syndrome was performed by Next Generation Sequencing. RAS and BRAF mutation analyses were performed using qPCR diagnostic kits. Epidemiology and risk factors, prevention and early detection programs, as well as treatment options and scientific advances have been described in detail. Out of 103 patients who underwent germline testing for Lynch syndrome, 19 (18.4%) showed a mutation in MMR genes with pathogenic or likely pathogenic significance and 8 (7.8%) in other CRC-associated genes (APC, CHEK2, MUTYH). Of 6369 tested patients, 50.43% had a mutation in KRAS or NRAS genes, while 9.54% had the V600 mutation in the BRAF gene. Although significant improvements in CRC management have occurred globally in recent years, a strategic approach leading to population-based systemic solutions is required. The high incidence of young-onset CRC and the growing elderly population due to a rise in life expectancy will be especially important factors for countries with limited healthcare resources like Serbia.

BRAF and NRAS Mutations and the Association with Prognosis of Acral Lentiginous and Nodular Melanomas in Indonesia.

Melanomas are rare yet the most aggressive skin cancer among Asians. Clinical presentation, risk factors, and the underlying molecular mechanisms are strikingly different from cutaneous melanoma in Caucasians. Mutation patterns of BRAF and NRAS genes were examined from DNAs derived from primary melanoma tumor tissues (fresh tissues or formalin-fixed paraffin-embedded samples) using pyrosequencing. A total of 63 patients consisting of acral lentiginous melanoma (N=22, 34.9%) and nodular melanoma (N=41, 65.1%) were included in this study. Most patients were diagnosed at Stage III-IV (N=49, 77.8%), Breslow thickness more than 4 mm (N=51, 80.9%), presence of ulceration (N=35, 55.6%), diameter larger than 6 mm (N=61, 96.8%), regional node infiltration (N=41, 77.8%). BRAF and NRAS mutations were found in 28 (44.4%) and 8 (12.7%), respectively. BRAF and NRAS mutations were significantly associated with older melanoma patients (OR = 6.075, 95%CI = 2.013-18.333 dan OR = 13.263, 95%CI = 1.518-115.901, respectively). BRAF mutations were associated with lower overall survival (Median survivals were 16.5 vs 31.4 months, Log-rank test P=0.001). NRAS mutations were not significantly associated with lower overall survival. In this study, melanoma patients are largely diagnosed at the late stages with ulceration and involvement of regional lymph nodes. BRAF mutations are associated with lower survival of cutaneous melanoma patients.

The RAS/BRAF genes status in patients with colorectal cancer (review)

Colorectal cancer (CRC) is the third in prevalence among oncological diseases worldwide and second in the structure of oncological mortality. Genetic assessment of CRC is a necessary stage during selecting further treatment for patients. Many studies demonstrate a diverse distribution of mutations in the KRAS, NRAS, and BRAF genes in CRC. A critical literature review was conducted in order to systematize data on the mutational profile and genetic heterogeneity of these driver mutations in Russian patients with CRC. Articles were searched for in open databases. Totally 17 Russian studies and 3 English meta-analyses were analyzed for comparison with Russian data. Mutations in the KRAS, NRAS, and BRAF genes, according to Russian and international studies, are found in 40 %, 4 %, and 7 % in CRC patients, respectively. The frequency and specific localization of mutations may depend on the geographical location and nationality of the cohort. High intertumoral and intratumoral heterogeneity in CRC, especially in KRAS gene mutations, significantly influences the choice of further therapy and underscores the need for more detailed study of the mutational profile of the primary tumor, affected lymph nodes, and distant metastases. In Russia, several molecular genetic methods are used to determine somatic mutations in CRC with different sensitivity and specificity, the most common is real-time PCR. More accurate diagnostic methods include digital droplet PCR, Sanger sequencing, and next-generation sequencing, but each method has its limitations that must be considered when planning diagnostics and research. The promising directions in personalized oncology is the study of gene copy number variations, which may contribute to the development of new methods for treating CRC in the future. Despite the large number of studies, some aspects of the mutational profile of CRC in Russian studies remain poorly understood, which is why further research is needed on patients with colorectal cancer in Russia.

Associations of SEMA7A, SEMA4D, ADAMTS10, and ADAM8 with KRAS, NRAS, BRAF, PIK3CA, and AKT Gene Mutations, Microsatellite Instability Status, and Cytokine Expression in Colorectal Cancer Tissue.

Semaphorins (SEMAs), ADAM, and ADAMTS family members are implicated in various cancer progression events within the tumor microenvironment across different cancers. In this study, we aimed to evaluate the expression of SEMA7A, SEMA4D, ADAM8, and ADAMTS10 in colorectal cancer (CRC) in relation to the mutational landscape of KRAS, NRAS, BRAF, PIK3CA, and AKT genes, microsatellite instability (MSI) status, and clinicopathological features. We also examined the associations between the expression of these proteins and selected cytokines, chemokines, and growth factors, assessed using a multiplex assay. Protein concentrations were quantified using ELISA in CRC tumors and tumor-free surgical margin tissue homogenates. Gene mutations were evaluated via RT-PCR, and MSI status was determined using immunohistochemistry (IHC). GSEA and statistical analyses were performed using R Studio. We observed a significantly elevated expression of SEMA7A in BRAF-mutant CRC tumors and an overexpression of ADAM8 in KRAS 12/13-mutant tumors. The expression of ADAMTS10 was decreased in PIK3CA-mutant CRC tumors. No significant differences in the expression of the examined proteins were observed based on MSI status. The SEMA7A and SEMA4D expressions were correlated with the expression of numerous cytokines associated with various immune processes. The potential immunomodulatory functions of these molecules and their suitability as therapeutic targets require further investigation.

Loss of Heterozygosity and Mutations in the RAS-ERK Pathway Genes in Tumor Cells of Various Loci in Multiple Myeloma.

Multiple myeloma (MM) is a disease characterized by spatiotemporal heterogeneity of tumor clones. Different genetic aberrations can be observed simultaneously in tumor cells from different loci, and as the disease progresses, new subclones may appear. The role of liquid biopsy, which is based on the analysis of tumor DNA circulating in the blood plasma, continues to be explored in MM. Here, we present an analysis of the STR profiles and mutation status of the KRAS, NRAS, and BRAF genes, evaluated in plasma free circulating tumor DNA (ctDNA), CD138+ bone marrow cells, and plasmacytomas. The prospective single-center study included 97 patients, with a median age of 55 years. Of these, 94 had newly diagnosed symptomatic MM, and three had primary plasma cell leukemia. It should be noted that if mutations were detected only in ctDNA, "non-classical" codons were more often affected. A variety of adverse laboratory and clinical factors have been associated with the detection of rare KRAS or NRAS gene mutations in bone marrow or ctDNA, suggesting that these mutations may be factors of an unfavorable prognosis for MM. Liquid biopsy studies provide undeniable fundamental information about tumor heterogeneity and clonal evolution in MM. Moreover, we focus on using liquid biopsy to identify new high-risk factors for MM.

Clinical features and prognostic factors of advanced myelodysplastic syndromes in children

Objective: To investigate the clinical features and prognostic factors of advanced myelodysplastic syndromes (MDS) in children. Methods: Clinical data of children diagnosed with advanced MDS in the Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, between September 2009 and April 2022 were retrospectively collected. Follow-up assessments were performed through telephone interviews and the review of medical records until May 1, 2023. The clinical features of children with advanced MDS were summarized by analyzing chromosomal karyotype tests, second-generation gene sequencing results. Multivariate Cox regression analysis was used to investigate the prognostic factors of advanced MDS in children. Results: A total of 69 children, comprising 49 males and 20 females, aged [M (Q1, Q3)] 8 (5, 10) years, were enrolled in the study. Sixty-seven cases underwent chromosomal karyotype testing, of which 42 cases (62.7%) had abnormal karyotypes, with monosomy 7 the most common in 17 cases (25.4%). Forty-three cases underwent next-generation sequencing, with mutations in the SETBP1, NRAS, PTPN11 and RUNX1 genes more common, identified in 12 cases (27.9%), 9 cases (20.9%), 8 cases(18.6%), and 8 cases(18.6%), respectively. The follow-up time [M (Q1, Q3)] was 26 (13, 56) months and the 5-year overall survival rate was 56%(95%CI: 44.4%-70.5%). The 5-year overall survival rate for children who underwent hematopoietic stem cell transplantation (HSCT) was higher than that of children who did not undergo HSCT (73.9% vs 29.1%, P<0.001). HSCT (HR=0.118, 95%CI: 0.037-0.372, P<0.001) was a protective factor for the overall survival rate of children with advanced MDS. Serum ferritin level>356.3 μg/L (HR=6.497, 95%CI: 2.068-20.415, P=0.001) and moderate to severe splenomegaly (HR=4.075, 95%CI: 1.174-14.141, P=0.027) were risk factors for the overall survival rate of children with advanced MDS. Conclusions: Monosomy 7 was the most common abnormal karyotype and SETBP1 was the gene that had the highest mutation frequency in children with advanced MDS. HSCT, increased ferritin and moderate to severe splenomegaly are prognostic factors influencing the overall survival rate of children with advanced MDS.

Diagnosis of metastatic melanoma by fine-needle aspiration biopsy and primary melanoma by using the skin and mucousa imprints

BACKGROUND: Determination of the cytogenetic origin and morphological type of metastatic or primary tumor determines the prescription of therapy and affects the efficiency of patient’s treatment. Currently, a number of publications evaluate the potential of cytological diagnostics of melanoma. AIM: To evaluate the significance of diagnosing metastatic melanoma by fine-needle aspiration biopsy and primary melanoma by using the skin and mucosa imprints. METHODS: In a retrospective study, a comparative analysis of cytological melanoma samples, in comparison with clinical and anamnestic information and the results of histological, immunohistochemical, and molecular genetic studies, was carried out. Information about 109 patients from the cancer registry of the Altai Regional Oncology Dispensary (Barnaul, Russia) for 2022 was used in the study. Traditional and liquid-based methods for preparing samples were used. The samples were stained using Pappenheim and Papanicolaou methods. In some observations, cytological material was used for molecular genetic studies. Using the cancer registry data of the dispensary, the results of histological and of molecular genetic studies, and a final conclusion was given about each patient. RESULTS: Fine-needle aspiration biopsy was carried out in 80 patients. Tumor smears were obtained from 29 patients. The cytological diagnose “melanoma” was consistent with the data of histological and immunohistochemical studies (p 0.001) for all 109 patients. Melanoma was diagnosed for the first time in 66 (60.5%) patients. In other cases, the process of progression was noted within the period from one year to 20 years. Epidermal melanoma was noted in 101 (92.7%) cases, including 9 patients with acral melanoma, and 2 cases with localization on the vulva. Melanomas of mucosa were found in 5 cases (4.5%): in the rectum and anal canal, vagina, and in 2 cases on the hard palate. Non-epidermal (uveal) melanomas metastases were diagnosed in liver by fine-needle aspiration biopsy in 3 patients (2.8%). Based on the cellular composition, epithelioid cell melanoma was determined in 81 (74.3%) patients, mixed cell in 14 (12.8%) cases, spindle cell in 9 (8.2%), pigmentless in 2 (1.8%) cases and nevoid melanoma in 1 (0.9%) case. The mutation status was determined in 96 patients (88.1%). Of these, in 8 patients it was determined using cytological material. In epidermal melanomas, mutations in codon 600 of exon 15 of the BRAF gene were found in 43 (44.8%) patients, including 2 cases of acral melanoma. Mutations V600K, V600E/Ec were found in one patient each. In mucousal melanomas: Q61R mutation was found in exon 3 of the NRAS gene (vaginal melanoma), G12C mutation was identified in exon 2 of the NRAS gene (anal canal melanoma). In uveal melanomas, the assessed mutations were absent (it is necessary to determine mutation is GNAQ11 and BAP1 genes). CONCLUSION: Cytological diagnosis of melanoma by fine-needle aspiration biopsies and imprints from the tumor mass is a highly informative method that allows diagnosing melanoma and verifying the tumor subtype. The obtained results indicate tumor heterogeneity and differences in mutational status depending on the location of melanomas. The molecular classification of melanoma is important when choosing individualized therapy.

Мутации в генах KRAS/NRAS и эффективность индукционной терапии по трехкомпонентным схемам на основе бортезомиба у пациентов с впервые диагностированной множественной миеломой

AIM. To identify the KRAS and NRAS gene mutations in patients with newly diagnosed multiple myeloma (ММ) and to classify them according to the depth of antitumor response to bortezomib-based triplet induction therapy. MATERIALS &amp; METHODS. The trial enrolled 89 patients with newly diagnosed MM prior to chemotherapy. Among them, there were 45 women and 44 men aged 30–82 years (median 58.5 years). ММ was diagnosed according to IMWG criteria (2014). Bone marrow (BM) plasma cells were isolated from the aspirate using gradient method with subsequent immunomagnetic CD138 marker selection. The KRAS and NRAS gene mutations in BM CD138+ cells were identified with Sanger sequencing method. The proteomic programs MutationTaster, Polyphen2, and FATHMM-XF were used for mutation analysis in the KRAS and NRAS genes. All patients received bortezomib-based triplet chemotherapy as first-line treatment. The response depth was assessed after completing 6 cycles of PAD and VCD regimens. Antitumor response was evaluated according to IMWG (2016) criteria. RESULTS. The mutation rate in the gene family RAS was 42 % (37/89). The analysis focused on the data from 33 patients with mutations detected and response identified after 6 cycles of treatment. In 22 out of 33 patients, deep response was not achieved, whereas 11 patients showed complete remission (CR) + very good partial remission (VGPR). In the group of patients without mutations in the gene family RAS, the response to therapy meeting the CR + VGPR criteria was 64 % (27/42). The differences appeared to be significant (p = 0.008). The clinical data and the evaluation of primary treatment outcomes provided the basis for distinguishing a group of 9 prognostically unfavorable mutations: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val. CONCLUSION. The mutations in KRAS and NRAS belonging to the gene family RAS had a negative effect on the efficacy of the bortezomib-based triplet induction therapy. Mutation variants in the RAS family genes differed in prognostic significance. The analysis results helped to identify the mutation variants associated with the worse response to therapy: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val.

Circulating tumor DNA analysis in patients with BRAF-mutated metastatic cutaneous melanoma treated with BRAF and MEK inhibitors: Analysis of the OPTIMEL study.

9541 Background: BRAF and MEK kinase inhibitors (KI) are indicated in combination for the treatment of adult patients with unresectable or metastatic melanoma (MM) harboring a BRAF V600 mutation. However, approximately half of the patients develop resistance to the treatment within a year. Circulating tumor DNA (ctDNA) has demonstrated its prognostic value for MM patients receiving specific treatment, offering potential assistance in patient follow-up. We present here the results of the prospective interventional study OPTIMEL (NCT03416933) that was conducted to investigate the relevance of ctDNA in predicting outcomes for MM patients undergoing BRAF and MEK inhibitors treatment. Methods: Thirty-five patients with histologically proven advanced cutaneous MM harboring BRAF mutations, either in a metastatic stage or cases ineligible for surgical intervention were enrolled in the study. All participants underwent treatment with BRAF and MEK inhibitors. Blood samples were systematically obtained on days 0, 15, 30, 90, 180, 270, and 365, or at the occurrence of disease progression. The assessment of mutations in the BRAF and NRAS genes in all plasma samples was conducted using the Idylla system (Biocartis, Mechelen, Belgium). Results: Out of the 35 patients initially enrolled in the study, 4 were excluded for not meeting the inclusion criteria, leaving 31 patients for the final analysis. At baseline, BRAF mutation was identified in 18 patients (58%), while it was not detected in 13 patients (42%). The presence of BRAF mutation at baseline was associated with a significantly lower progression-free survival (PFS) (HR = 3.42; CI95% [1.19;9.82]; p = 0.022). During the follow-up, BRAF mutation was detected in the plasma of 19 out of 31 patients. A BRAF variant allele frequency (vaf) below 2.3% was strongly linked to an objective response to treatment (p&lt;0.001). The absence of BRAF variant detection during follow-up was significantly associated with better outcome (PFS 4.01 vs 12.0 months, HR = 28.5; CI95% [7.75;105.0]; p &lt; 0.001). Moreover, PFS significantly deteriorated when BRAF vaf exceeded 4.5% (3.15 vs 12.0 months, HR = 112; CI95% [13.8;913.0]; p &lt; 0.001). Lastly, the identification of NRAS mutation in plasma during follow-up was linked to treatment resistance and a worsened PFS (4.84 vs 10.9 months, HR = 11.6; CI95% [2.76;48.5]; p&lt;0.001). Conclusions: This report presents the initial analysis of the OPTIMEL study. The variant allele frequency of BRAF mutations in circulating tumor DNA is indicative of the objective response to BRAF and MEK inhibitors, as well as PFS. These findings could potentially influence our approach to managing patients with MM harboring a BRAF mutation. Clinical trial information: NCT03416933 .

Multicenter clinical trial of DNA markers for non-invasive differential diagnosis of benign and malignant melanocytic lesions.

e21550 Background: The differentiation between benign melanocytic neoplasms (nevi) and malignant melanocytic neoplasms (melanoma) presents a significant diagnostic challenge, necessitating the development of accurate and reliable diagnostic markers. This study aimed to evaluate the analytical characteristics of selected DNA markers for their efficacy in differentiating these conditions. Methods: A prospective analysis was conducted on sample groups obtained from participants of the multicenter clinical study, MoleMed. The study included 112 histological samples from 100 patients and a corresponding number of cytological samples, collected with the aim of evaluating DNA markers' sensitivity and specificity. DNA extraction followed a previously developed protocol, with concentration and quality assessed through microchip electrophoresis and real-time PCR. Molecular testing targeted mutations in "hotspots" of the BRAF and NRAS genes, as well as promoter mutations in the TERT gene, employing mutation-specific PCR amplification and Sanger sequencing for verification. Results: DNA extraction was successfully performed for 101 cytological samples, with concentrations ranging from &lt; 0.001 ng/µl to 5,508 ng/µl; the average concentration was 106.52 ng/µl, and the median was 2.37 ng/µl. BRAF mutations were found in 28 samples, with the mutation c.1799T &gt; A (p.Val600Glu, V600E) in 21 samples, c.1798_1799GT &gt; AA (p.Val600Lys, V600K) in 6 samples, and c.1799_1800TG &gt; AT (p.Val600Asp, V600D) in 1 sample. NRAS mutations were detected in 11 samples, including c.181C &gt; A (p.Gln61Lys, Q61K) in 6 samples, c.182A &gt; G (p.Gln61Arg, Q61R) in 3 samples, and c.182A &gt; T (p.Gln61Leu, Q61L) in 2 samples. No cases of simultaneous presence of more than one mutation in BRAF and NRAS "hotspots" within the same cytological sample were observed. DNA profiling of cytological material revealed signs of malignant neoplasms (MN) in 49 samples and no signs of MN in 52 samples. TERT mutations c.-124C &gt; T were detected in 21 samples, c.-146C &gt; T in 31 samples (including "borderline" cases), and c.-139_-138CC &gt; TT in 4 samples. Six samples exhibited more than one mutation, highlighting the complexity of genomic alterations in these neoplasms. Conclusions: The PCR analysis of the five DNA markers on both histological and cytological samples of human skin melanomas and nevi demonstrates promising analytical characteristics, with sensitivity and specificity exceeding 70% for all tested scenarios. These results highlight the potential utility of these DNA markers in the differential diagnosis of melanocytic neoplasms, offering a reliable, non-invasive diagnostic tool that complements traditional histopathological examination. Clinical trial information: NCT04353050 .