Resection of metastases in patients with BRAF mutated metastatic colon cancer: results of a multicenter retrospective study

Introduction: local treatment of metastases is an integral part of colon cancer treatment. However, there is not enough data on the efficacy of surgical resection of metastases in patients with a BRAF gene mutation to recom‑mend this approach in routine practice. We initiated a retrospective multicenter study to assess the incidence of BRAF gene mutations in patients with metastatic colon cancer and to study the efficacy of metastasectomy in this group of patients.Materials and methods: we selected all patients who underwent surgical resection of metastases in various sites from the database of patients with BRAF gene mutations created as a result of a multicenter retrospective study with participation of 7 clinics in the Russian Federation. All 57 patients with RAS gene mutations and 43 patients with wild‑type RAS and BRAF genes who also underwent surgical resection of metastases at any stage of treatment were selected from the register of the Chemotherapy Department No. 2 of the NMRC of Oncology named after N. N. Blokhin for comparative analysis. Disease‑free survival and overall survival were used as primary efficacy criteria.Results: we found 26 patients with BRAF gene mutations who underwent surgical resection of metastases. When comparing disease‑free survival, the worst median was achieved in the group of patients with BRAF gene mutations: 7 months versus 14 months in patients with RAS gene mutations (HR 0.4, 94 % CI 0.23–0.7, P = 0.006); median disease‑free survival was not achieved in the wild‑type RAS and BRAF group (HR 0.2, 95 % CI 0.11–0.45, P <0.001).The median overall survival in the BRAF gene mutation group was 26 months versus 38 months in the RAS gene mutations group (HR 0.8, 95 % CI 0.33–1.98, P = 0.6) and 49 months in the wtRAS/wtBRAF group (RR 0.46, 95 % CI 0.17–1.24, P = 0.1). Resection of recurrent tumors in patients with metastases in retroperitoneal lymph nodes was associated with extremely low disease‑free survival (2 months); at the same time, disease‑free survival was 7 months after resection of isolated metastases in the liver and 8 months for metastases in the peritoneum.Conclusion: prognosis of patients with a BRAF gene mutation after surgical resection of metastases is worse than in patients with a different mutation phenotype. Nevertheless, literature data, as well as the results of our study, confirm the possibility of performing metastasectomy with careful selection of patients.

Cholangiocarcinogenesis and targeted therapy for cholangiocarcinoma

Cholangiocarcinoma includes a highly heterogeneous group of malignant tumors of the biliary tract, developing from the epithelium of the intra- and extrahepatic bile ducts. The incidence of cholangiocarcinoma is growing worldwide and currently accounts for about 15% of all primary neoplastic diseases of the liver and up to 3% of malignant neoplasms of the gastrointestinal tract. The asymptomatic course of these tumors in combination with a very aggressive course and low sensitivity to cytotoxic therapy contributes to a fairly high mortality rate from this disease, amounting to up to 2% in the structure of cancer mortality in the world. The high heterogeneity of cholangiocarcinoma at the genomic, epigenetic and molecular levels significantly reduces the effectiveness of the available treatments. In recent decades, new diagnostic tools and treatment methods have been developed to improve the results of treatment of patients with cholangiocarcinoma. The prevalence of BRAF gene mutations is associated with the occurrence of various tumors, including cholangiocarcinoma. Currently, more than 30 mutations in the BRAF gene with oncogenic potential have been registered. Basically, the V600 codon is affected, an amino acid change occurs, which ultimately leads to the fact that the BRAF protein becomes constantly active, even in the absence of growth factors. Dabrafenib is an inhibitor of the BRAF protein, which is permanently overactive in mutated cells. The use of dabrafenib as monotherapy leads to the development of resistance after 6–7 months. Dabrafenib is used with trametinib, a MEK inhibitor that also blocks the Ras-Raf-MEK-MAPK kinase pathway, to prevent the resistance.

Epigenetic modification and BRAF gene mutation in thyroid carcinoma

Thyroid cancer remains the most prevailing endocrine malignancy, and a progressively increasing incidence rate has been observed in recent years, with 95% of thyroid cancer represented by differentiated thyroid carcinomas. The genetics and epigenetics of thyroid cancer are gradually increasing, and gene mutations and methylation changes play an important roles in its occurrence and development. Although the role of RAS and BRAF mutations in thyroid cancer have been partially clarified,but the pathogenesis and molecular mechanisms of thyroid cancer remain to be elucidated. Epigenetic modification refer to genetic modification that does not change the DNA sequence of a gene but causes heritable phenotypic changes in its expression. Epigenetic modification mainly includes four aspects: DNA methylation, chromatin remodelling, noncoding RNA regulation, and histone modification. This article reviews the importance of thyroid cancer epigenetic modification and BRAF gene mutation in the treatment of thyroid cancer.

Joint Effects of Co-Exposure to Polycyclic Aromatic Hydrocarbons Exposure and Smoking with XPC Polymorphisms on Damage in Exon 2 of KRAS Gene Among Young Coke-Oven Workers

Background Genetic polymorphisms may contribute to variable individual susceptibility to DNA damage induced by co-exposure to polycyclic aromatic hydrocarbons (PAHs) and smoking. Here, we evaluate the joint effects of co-exposure to PAHs and smoking with polymorphisms in XPC, alone or combined, on damage in exons. Methods 288 healthy male coke-oven workers were enrolled into this study, urinary 1-hydroxypyrene (1-OH-pyr), as an indirect and sensitive indicator of PAHs exposure, was detected. Base modification in exons of KRAS and BRAF gene, and polymorphisms of XPC were determined by real-time PCR in plasma samples. Results We observed urinary 1-OH-pyr was positively related to damage index of exon 2 of KRAS gene (KRAS-2) and damage index of exon 15 of BRAF gene (BRAF-15) (both p < 0.001), and KRAS-2 and BRAF-15 were both significantly associated with increased urinary 1-OH-pyr (both p trend < 0.001). A stratified analysis found that urinary 1-OH-pyr was found to be significantly associated with KRAS-2 in both smokers (p < 0.001) and nonsmokers (p < 0.05), while urinary 1-OH-pyr was significantly associated with BRAF-15 only in smokers (p < 0.001). Additionally, rs2228001 in the XPC gene was positively associated with KRAS-2 (p trend < 0.001), and the high levels of 1-OH-pyr were linearly associated with KRAS-2 only in rs2228001 GG+GT genotype carriers (p < 0.001); However, another SNP rs3731055 in the XPC gene was associated with lower KRAS-2 (p trend = 0.006), and the high levels of 1-OH-pyr were linearly associated with KRAS-2 only in rs3731055 GG genotype carriers (p < 0.001). Moreover, KRAS-2 significantly increased with high levels of 1-OH-pyr exposure, smoking, rs2228001 G-allele, and rs3731055 GG homozygote genotype, and we found the most severe KRAS-2 among subjects carrying all 4 of the above risk factors. Conclusions Our findings indicated that the co-exposure effect of PAHs and cigarette smoking could increase the risk of damage in exon 2 of KRAS by a mechanism partly involving XPC polymorphisms.

BRAF Fusions in Histiocytic Disorders: Frequency and Clinical Characteristics

Background: Activation of extracellular-signal regulated kinase (ERK) is almost universal in histiocytic disorders and is driven by genomic alterations in the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K) pathways. While BRAFV600E mutations are well described in histiocytic disorders, less is known about BRAF fusions. The goal of our study is to describe the frequency and clinical characteristics of patients harboring BRAF fusions. Methods: We included patients with a diagnosis of histiocytic disorder seen at our institution from November 2016-June 2021. Only those who had adequate BRAF testing were included. The following were considered adequate BRAF testing: 1) unequivocally positive BRAF V600E immunostain with or without molecular confirmation; 2) successful multigene next generation sequencing (mostly Tempus â or FoundationOne â) if BRAF V600Eimmunostain was equivocal or negative. We also searched the literature for similar cases and compared BRAF fusions found in histiocytic disorders to those found in solid tumors and other hematologic conditions. Results: One hundred and twenty-six patients were included in this study. BRAF fusions were detected in 7 (6%) patients. The median follow-up for these patients was 1.4 years (95% CI: 0.4-not reached). The frequency according to disease subtypes is as follows: Erdheim-Chester disease (ECD; 2/46 [4%]), Langerhans cell histiocytosis (LCH; 1/41 [2%]), adult or juvenile xanthogranuloma (AXG/JXG; 4/7 [57%]), and histiocytic or Langerhans cell sarcoma (HS/LCS; 0/9 [0%]). The median age at diagnosis for our study cohort was 34 years (range, 7-81 years) and 5 were females. The clinical characteristics and nature of BRAF fusions of our 7 patients plus additional 13 cases from the literature are shown in Table 1. For the final cohort of 20 patients with BRAF-fusions, the median age was 16 years (range, 0.5-81 years) and most (56%) were females. The distribution by histiocytic subtypes is as follows: AXG/JXG (45%), LCH (40%), ECD (10%), HS/LCS (5%). Two patients received cobimetinib as frontline treatment in our cohort and achieved partial responses. Of these two patients, one completed 12 cycles (12 months) of cobimetinib and had a sustained PR while the other patient received 2 cycles before having to hold therapy due to intolerable adverse effects despite dose reduction. From literature review, another patient (LR-12) received cobimetinib as second line treatment and achieved complete response. We found 15 unique BRAF gene fusions with 4 being recurrent. Except for AGAP3-BRAF, none of these BRAF fusions in histiocytosis has been reported in solid tumors or hematologic conditions to date. Conclusions: BRAF gene fusions occur rarely (&lt;5%) in histiocytic disorders. The only exception is AXG/JXG where more than half of the patients harbor BRAF gene fusions. These gene fusions are mostly distinct to histiocytosis and not found in solid tumors or other hematologic conditions. Figure 1 Figure 1. Disclosures Bennani: Purdue Pharma: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Vividion: Other: Advisory Board; Kymera: Other: Advisory Board; Verastem: Other: Advisory Board.

DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes.

BRAF Gene Mutation (V600E) in Aspiration Cytology of Patients With Suspected Papillary Thyroid Carcinoma

This study was attempted to investigate the prevalence of BRAF gene mutation (V600E) in aspiration cytology of patients with suspected papillary thyroid carcinoma (PTC). Seventy-six patients suspected of having PTC who were referred for fine-needle aspiration (FNA) biopsy were included in this cross-sectional study. Ultrasound-guided FNA was taken from the thyroid masses, and samples were sent for cytologic evaluation. Simultaneously, the samples were sent to a genetic laboratory to check the status of BRAFV600E mutation. Patients with FNA positive for PTC were assigned in one group, and those with FNA negative for PTC were assigned to another group. Cytological and molecular results were compared with those of histopathology and sonography. The results showed that the prevalence of the BRAF gene (V600E) mutation in our study was 21.1% (16 out of 76 patients). In addition, the results showed a significant relationship between gene mutation and pathologic findings so that the highest gene mutation was significantly detected in patients with FNA positive for PTC (P=0.001). Also, our results showed a significant relationship between gene mutation and some sonographic findings (calcification, P=0.004) and no significant relation in the other sonographic findings (hypoechoic changes, P=1.12 and regular changes, P=0.194). According to the results of the present study, BRAF mutation (V600E) can be an effective indicator for definitive diagnosis and primary treatment of PTC in suspected cases.

New factors of pterygium occurrence and recurrence after surgical treatment

Background. Occurrence and recurrence of pterygium after surgical treatment are still urgent problems of modern ophthalmology. The purpose was to determine the main factors leading to the occurrence and recurrence of pterygium after surgical treatment. Materials and methods. We observed 203 patients (232 eyes) with pterygium. There were 108 men and 95 women. The age of patients ranged from 35 to 65 years. The duration of the disease is 2.5–2.7 years. Patients underwent surgical treatment for pterygium by the McReynolds’ method forming a cul-de-sac, by the Artl method with its incision, and the McReynolds and Artl methods using 0.02% solution of mitomycin C. The patients were examined in 3, 6 months, and one year after surgery. The statistical analysis of the results of clinical trials was performed using the software package SPSS 11.0, MedStat (Lyakh Y.E., Guryanov V.G., 2004–2012), MedCalc (MedCalc Software bvba, 1993–2013). Results. The results of clinical studies have found that the frequency of pterygium recurrence after traditional surgical interventions by the McReynolds method, the McReynolds method using 0.02% solution of mitomycin C, by the traditional Arlt method, the Arlt me­thod using 0.02% solution of mitomycin C in 3 months was 20, 17, 15, and 8.7 %, respectively; in 6 months — 29.5, 27, 27, and 26 %, respectively; after 1 year — 26.5, 26.5, 30.6, and 37 %, respectively. Long-term recurrence of pterygium did not differ significantly (p < 0.05). Viral infection was found in pterygium tissue in 50.9 % of cases, including herpesviruses (33.6 %) and human papillomavirus (HPV, 34.0 %). Among herpesviruses, herpes simplex virus (HSV) was found in 15.1 % of cases, cytomegalovirus (CMV) in 7.3 %, and Epstein-Barr virus (EBV) in 11.2 % of cases. The regression analysis showed an association of HSV with stage II and stage III pterygium (p < 0.05). No such dependencies were found for CMV and EBV. There was a relationship of HPV with the I–III stages of pterygium (p < 0.01). There were types 6 (34.2 %), 11 (24.1 %), 16 (21.5 %), and 18 (20.2 %) among HPV. Types 6 and 11 were mainly detected at the first stages and were not detected in stage IV; types 16 and 18 were not detected in stage I (p = 7.9e-04). Among other types, only HPV6 was associated with the I and III stages of pterygium (p < 0.05). Double mixed infection was detec­ted in 32.1 % of patients. The most common was the combination of HSV and HPV (30.6 % of all cases of mixed infection), three viruses — in 2.5 % of cases in the combination of HSV + CMV + HPV and HSV + EBV + HPV. It was first established that the V600E mutation of the BRAF gene was detected in 35.3 % of cases of pterygium. No significant difference in mutation frequency depending on the sex and age of patients was found. The frequency of mutation by stages increased from 21.3 % at stage I to 57.7 % at stage IV (p = 0.0003). The regression analysis showed a strong progressive association with the presence of the BRAF gene V600E mutation with the pterygium stage and, therefore, its progression. Conclusions. Thus, it was proved that the presence of HSV, HPV, and mutation V600E of the BRAF gene significantly influenced the occurrence and progression of pterygium after surgical treatment.

Evaluation of BRAF Gene Status in Gliomas

Background: Development of different molecular markers has given a new insight in the glioma management. KIAA1549-BRAF gene fusion has a diagnostic and prognostic significance. Aim: The aim of this study was to determine the KIAA1549-BRAF gene fusion in glioma and their correlation with various clinical parameters. Material and Methods: Forty cases of glioma were studied for KIAA1549-BRAF gene fusion by reverse transcription-PCR (RT-PCR). Results: Overall, KIAA1549-BRAF gene fusion was found in 22% (9/40) cases of glioma. Children had higher KIAA1549-BRAF fusion (72%; 8/11) as compared to adults (10%; 3/29) and this difference was statistically significant. Cerebellar location of tumor was significantly associated with KIAA1549-BRAF fusion. KIAA1549-BRAF fusion was highest in pilocytic astrocytoma (89%), and this difference was statistically significant. Statistically significant difference was noted between KIAA1549-BRAF fusion expression and WHO grade I glioma. Conclusion: Overall, KIAA1549-BRAF gene fusion was found in 22% (9/40) cases of glioma. Childhood age, pilocytic astrocytoma histology, cerebellar location and WHO grade I tumor were significantly associated with KIAA1549-BRAF gene fusion.