BRAF Gene Mutations Research Articles

Analysis of Risk Factors and Risk Prediction for Cervical Lymph Node Metastasis in Thyroid Papillary Carcinoma.

To analyze the risk factors of cervical lymph node metastasis (LNM) of thyroid papillary carcinoma (PTC) and construct the prediction model. Clinical data of 1105 patients with pathologically confirmed PTC in our hospital from February 2019 to May 2024 were retrospectively analyzed, and randomly divided into a training set and validation set according to the proportion of 7:3. With cervical central LNM (CLNM) and lateral LNM (LLNM) as outcome variables respectively, ultrasound characteristics were analyzed and C-TIRADS scores were performed Combined with the general situation of the patient, preoperative serum thyroglobulin (Tg) level, BRAFV600E (hereinafter referred to as BRAF) gene mutation and other characteristics of the patient, analysis was conducted to determine the independent risk factors for cervical CLNM and LLNM of PTC, and establish Nomogram prediction models. The test data set is used to validate the model. The area under the ROC curve (AUC) and the decision curve analysis (DCA) were used to evaluate the prediction efficiency of the model. The analysis shows that male, age < 55 years old, tumor diameter ≥ 1 cm, capsular invasion, positive serum thyroglobulin (Tg), BRAF gene mutation type and C-TIRADS score are independent risk factors for cervical CLNM in PTC (P < 0.05). Tumor diameter ≥ 1 cm, capsular invasion, tumor located at the upper pole and presence of CLNM are independent risk factors for LLNM in PTC. Based on the above risk factors, Nomogram prediction models for CLNM and LLNM are constructed respectively. The AUC of the CLNM prediction model is 91.5%. LLNM model is 96.1%. Ultrasound indicators, C-TIRADS score combined with BRAF gene status, Tg and clinical indicators of patients have important value in predicting cervical CLNM and LLNM in PTC. The Nomogram prediction models constructed based on the above indicators can effectively predict the risk of LNM in PTC.

Expert consensus on BRAF inhibitors in the treatment of malignant solid tumors (2024 edition)

The global aging population is leading to an increasing incidence of cancer, exacerbating the global burden of cancer. By 2040, the total number of cancer patients worldwide is projected to reach 28 million. With advancements in tumor molecular biology research and the widespread application of next-generation sequencing technology, precision treatment for cancer has made significant progress in clinical settings. Selective targeting of the Braf gene has emerged as one of the early successful cases of precision medicine for tumors. Braf gene mutations can result in unrestricted activation of downstream kinases in the mitogen-activated protein kinase (MAPK) cell signaling pathway, promoting rapid proliferation of tumor cells. BRAF inhibitors, either as monotherapy or in combination with MEK inhibitor, have been approved for various cancers, including melanoma, non-small cell lung cancer, thyroid cancer, colorectal cancer and glioma, among others. Clinical studies related to BRAF inhibitors are continuously exploring new applications. However, there is currently no consensus on the use of BRAF inhibitors in the diagnosis and treatment of multiple solid cancers in China. This article integrates clinical evidence of Braf mutations in multiple solid cancers to establish an expert consensus on the diagnosis and treatment of malignant solid cancers with Braf gene mutations. The goal is to promote and guide the standardized application of BRAF inhibitors.

Multi-gene mutations in ultrasound-guided fine-needle aspiration specimens of thyroid micronodules and diagnostic value in thyroid microcarcinomas using next-generation sequencing

Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [M (Q1, Q3)] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P>0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95%CI) of 0.857 (0.789-0.925). For nodules classified as Bethesda Ⅲ-Ⅴ, the sensitivity and specificity were 63.0% and 100.0%, respectively, with an AUC (95%CI) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

Một số bất thường gen ở bệnh nhân ung thư phổi không tế bào nhỏ tại Bệnh viện Đại học Y Hà Nội phát hiện bằng phương pháp giải trình tự gen thế hệ mới

This study aimed to evaluate the rate of genetic mutations in patients with non- small-cell lung cancer by Next-Generation Sequencing at Hanoi Medical university hospital from 2022 to 2023 and to assess the relationship between genetic mutations and pathological characteristics in non-small-cell lung cancer. In 90 non-small-cell lung cancer patients in the study, we found EGFR gene mutations in 41.1% of cases; KRAS gene mutation in 11.1%; ALK gene fusion in 4.4%; PIK3CA gene mutation in 2.2%; BRAF gene mutation in 1.1%; MET gene mutation in 1.1% and ROS1 gene fusion in 1.1%. We found that EGFR mutations were more commonly detected in female, non-smokers and having adenocarcinoma while KRAS mutations were more commonly detected in male.

DICER1 Mutations Enable Rare Tumor Formation

Abstract Introduction/Objective DICER1 is a tumor suppressor gene that imparts a slight increase in tumor formation when mutated. The protein plays a role in processing miRNA that silences transcription. Germline mutations of DICER1 predispose to tumors in a known set of organs and rarely in other locations. However the presence of additional genetic aberrations further increases the likelihood of tumor formation. Acting as a compounding factor, DICER1 mutations can present a constantly shifting cancer phenotype of affected patients. Of particular interest here is the development of both Pineoblastoma and Malignant spindle cell sarcoma in a young patient. Sarcomas are estimated to make up only 10% of pediatric cancers and Pineal masses only 3%. Methods/Case Report This case involves an 8 year old male with a history of Pineoblastoma who originally presented with slowly worsening ataxia. A screening ultrasound on following visits identified an abdominal mass that was confirmed with computed tomography. Subsequent biopsies showed malignant spindle cells arranged in a herringbone pattern. Additionally these cells displayed high mitotic activity and hyperchromatic nuclei. Surprisingly, they also demonstrated extramedullary hematopoiesis. Multiple stains were performed however no significant conclusion could be made. The appearance of this tumor and its rarity prompted a request for a Complete Comprehensive Sarcoma Panel via Next Generation Sequencing. This confirmed the presence of pathologic BRAF and DICER1 genes. From that the diagnosis of DICER1 associated Malignant Spindle Cell Sarcoma was made. BRAF V600E is the most common genetic variant and is seen in multiple cancers but only seen in 1-3% of sarcomas. Pathologic variants of the DICER1 gene exist on exons 13 and 25. The presence of these exon mutations in some way affect DICER1 protein function and compound the effects of the mutated BRAF gene. This situation is unique as such neoplasms are rarely found in the anterior abdominal wall and showcases the effects of DICER1 on rare tumor phenotypes. Results (if a Case Study enter NA) NA Conclusion Continuing research into both DICER1 protein pathways is essential for isolating a diagnosis and guiding treatment. It would seem screening images and genetic testing would benefit pediatric or even adult patients with common and rare cancers. Extra effort made toward this would not be outside the norm as personalized medicine is gradually becoming the standard for cancer treatment.

Exploring immunotherapy efficacy in non-small cell lung cancer patients with BRAF mutations: a case series and literature review.

The use of immunotherapy in treatment of non-small cell lung cancer (NSCLC) patients with the BRAF gene mutations is an area of active research and is an item of clinical trials. While BRAF mutations are relatively infrequent in NSCLC patients, comprising approximately 1-3% of cases, the V600E substitution stands out as the most prevalent subtype of BRAF mutations. The presence of this mutation in cancer cells qualifies the patients for first-line therapy with BRAF and MEK inhibitors. This study aims to evaluate the efficacy of immunotherapy in NSCLC patients with BRAF mutations. We presented a series of seven NSCLC cases with BRAF mutations, four of whom received immunotherapy or chemoimmunotherapy. We observed benefit from immunotherapy in all patients, but its duration depended on comorbidities and the presence of brain metastases. Utilization of the next generation sequencing (NGS) technique causes high detection frequency of BRAF mutations (4.7% of patients), although mutations other than V600E may predominate (4 out of 7 patients). In patients receiving immune checkpoint inhibitors (ICIs)-based therapy, the median progression-free survival (PFS) was 17 months from the start of immunotherapy, the overall objective response rate (ORR) was 50%, and disease control was achieved in all patients. Immunotherapy can benefit NSCLC patients with BRAF mutations, though its efficacy is affected by comorbidities and brain metastases. The use of NGS enhances mutation detection, highlighting the need for personalized treatment approaches in NSCLC management. The varying responses to treatments among the patients emphasize the complexity of NSCLC management and the necessity for a personalized approach.

Follicular cell-derived thyroid carcinomas harboring novel genetic BRAFNON-V600E mutations: real-world data obtained using a multigene panel.

To assess the molecular profile of follicular cell-derived thyroid carcinomas (FCDTCs) and correlate the identified mutations with the clinical and pathological features of the affected patients. Cross-sectional study of tumor samples from 100 adult patients diagnosed with FCDTC between 2010 and 2019. The patients' clinical and pathological data were collected. Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumors using the ReliaPrep FFPE gDNA Miniprep System. Genotyping of target genomic regions (KRAS, NRAS, BRAF, EGFR, and PIK3CA) was performed using the AmpliSeq panel, while sequencing was performed on the iSeq 100 platform. The patients' mean age was 39 years. In all, 82% of the tumors were classic papillary thyroid carcinomas. Overall, 54 (54%) tumor samples yielded satisfactory results on next-generation sequencing (NGS), of which 31 harbored mutations. BRAF gene mutations were the most frequent, with the BRAF V600E mutation present in 10 tumors. Seven tumors had BRAF NON-V600E mutations not previously described in FCDTCs (G464E, G464R, G466E, S467L, G469E, G596D, and the T599Ifs*10 deletion) but described in other types of cancer (i.e., skin/melanoma, lung, colorectal, and others). One tumor had a previously reported BRAF A598V mutation. EGFR gene mutations were found in 16 (29%) and KRAS or NRAS alterations in 8 (14%) of the 54 tumors analyzed. We described herein seven non-hotspot/novel variants in the BRAF gene, highlighting their potential role in expanding our understanding of FCDTC genetics.

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.

Clinicopathologic Analysis of HPV-Related Primary Squamous Cell Carcinoma of the Thyroid.

This study aims to explore the clinical and pathologic characteristics of HPV-related primary thyroid squamous cell carcinoma (PSCCT), a rare tumor classified by WHO-5 as a subtype of anaplastic thyroid carcinoma (ATC). Clinical data, histomorphology, immunohistochemistry, HPV detection, and B-raf gene point mutations of 3 PSCCT cases were analyzed. Subsequent follow-up was conducted post-treatment. All 3 cases involved female patients aged between 60 and 76. Microscopic examination revealed squamous cell carcinoma in cases 1 and 3, whereas case 2 exhibited both squamous cell carcinoma and papillary thyroid carcinoma components. Immunohistochemistry demonstrated CK19, PAX8, and TTF1 expression in the papillary thyroid carcinoma component, and CK5/6, p63, p40, and PAX8 expression in the squamous cell carcinoma component. P16 exhibited diffuse positivity in both squamous cell carcinoma and classic papillary carcinoma. HPV analysis identified low-risk type 6 positivity in cases 1 and 3, while both squamous cell carcinoma and papillary carcinoma areas in case 2 were positive for HPV-33. B-raf gene mutation was exclusive to case 2. Diagnosis of PSCCT necessitates multidisciplinary assessment, incorporating clinical symptoms, imaging, histomorphology, and immunohistochemistry. This study, for the first time, reveals the presence of HPV DNA in both PTC and PSCCT, occurring concurrently but separately. Given the limited scope of 3 case reports, definitive conclusions cannot be drawn, warranting further investigation.

Locally advanced rectal cancer in a young adult affected with dyskeratosis congenita (Zinsser–Cole–Engman syndrome): a case report

BackgroundDyskeratosis congenita (DKC), also known as Zinsser–Cole–Engman syndrome, is a progressive genetic disease with a triad of reticulate skin pigmentation, nail dystrophy, and leukoplakia. Approximately 8–10% of patients with DKC develop malignancies, and cases of colorectal cancer with DKC in young people have been reported previously.Case presentationA 25-year-old man with DKC since approximately 10 years of age developed fever and lower abdominal discomfort. Diagnostic imaging revealed locally advanced rectal cancer with lymph node metastasis, direct invasion of the prostate, and pelvic abscess due to tumor microperforation (cT4bN2M0 cStage IIIC). Biopsy showed well to moderately differentiated ductal adenocarcinoma. Genetic testing was negative for RAS and BRAF gene mutations, and microsatellite instability (MSI) testing was also negative. After sigmoid colostomy, the patient was treated with total neoadjuvant therapy (TNT) with systemic chemotherapy (six courses of FOLFOX + panitumumab) followed by chemoradiation therapy (50.4 Gy with capecitabine). After TNT, the primary tumor and metastatic lymph nodes shrank. According to the findings of colonoscopy and magnetic resonance image (MRI), we diagnosed near complete response (near-CR) and decided to follow the patient without surgery by every 3 months re-evaluation. However, 5 months after TNT, tumor regrowth was detected on colonoscopy and imaging, and the patient underwent total pelvic exenteration. He developed paralytic ileus as a postoperative complication, and was discharged on the 38th postoperative day. Pathological examination revealed a residual tumor with invasion of the periprostatic tissue. There was no metastasis in the pararectal and lateral pelvic lymph nodes, but one extramural non-contiguous cancerous extension (tumor deposit) was observed (ypT4bN1cM0 ypStage IIIC). The patient has been free of recurrence for one year after surgery.ConclusionsDKC often develops into various tumors in the digestive system at an early age; therefore, appropriate surveillance may be required. In addition, considering that cancers in patients with DKC occur at a young age, fertility preservation and survivorship are also important, and adequate explanations and care should be provided to patients before and after treatment.

Gene mutation characteristics of clinical stage ⅠA lung adenocarcinoma and their relations with patients' long-term prognosis

Objective: To explore the gene mutation characteristics and the relationship between gene mutations and long-term prognosis in clinical stage ⅠA lung adenocarcinoma patients. Methods: A retrospective analysis was conducted on 63 clinical stage ⅠA lung adenocarcinoma patients who underwent surgical resection at the Cancer Hospital of the Chinese Academy of Medical Sciences from January 2007 to October 2012, with documented postoperative recurrence or metastasis, as well as those who had a follow-up duration of 10 years or more without recurrence or metastasis. Whole exome sequencing (WES) technology was used to analyze the gene mutation profiles in tumor tissues and univariate and multivariate Cox regression analysis were used to clarify the influencing factors for patient prognosis. Results: After long term follow-up, 13 out of the 63 patients (21%) experienced recurrence or metastasis. WES technology analysis revealed that the most common tumor related gene mutations occurred in epidermal growth factor receptor (EGFR), with a mutation rate of 65.1% (41/63), followed by tumor protein p53 (TP53), fatatypical cadherin 1 (FAT1), low density lipoprotein receptor-related protein 1B (LRP1B), mechanistic target of rapamycin (MTOR), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG), and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), with mutation rates of 30.2% (19/63), 20.6% (13/63), 15.9% (10/63), 15.9% (10/63), 15.9% (10/63), and 15.9% (10/63), respectively. Multivariate Cox regression analysis showed that PIK3CG mutations (HR=21.52, 95% CI: 3.19-145.01),smoothened (SMO) mutations (HR=35.28, 95% CI: 3.12-398.39), catenin beta 1 (CTNNB1) mutations (HR=332.86, 95% CI: 15.76-7 029.05), colony stimulating factor 1 receptor (CSF1R) mutations (HR=8 109.60, 95% CI: 114.19-575 955.17), and v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutations (HR=23.65, 95% CI: 1.86-300.43) were independent risk factors affecting the prognosis of clinical stage ⅠA lung adenocarcinoma patients. Conclusions: PIK3CG, SMO, CTNNB1, CSF1R, BRAF gene mutations are closely related to long-term recurrence or metastasis in clinical stage ⅠA lung adenocarcinoma. Patients with these gene mutations should be given closer clinical attention.

Real-World Cardiotoxicity in Metastatic Melanoma Patients Treated with Encorafenib and Binimetinib.

Modern therapies targeting the BRAF gene mutation in advanced melanoma have significantly improved patient outcomes but pose cardiovascular risks. This retrospective study in Eastern Denmark (2019-2022) assessed 108 melanoma patients treated with encorafenib and binimetinib. Patients were monitored for heart function using multigated acquisition (MUGA) scans. The study defined major cardiotoxicity as a decline in left ventricular ejection fraction (LVEF) by more than 10 percentage points to below 50%, and minor cardiotoxicity as a decrease in LVEF by more than 15 points but remaining above 50%. Results showed that 19 patients (18%) developed minor cardiotoxicity and were asymptomatic, while 7 (6%) experienced major cardiotoxicity, with two requiring intervention. Notably, no significant declines in LVEF were observed after six months of treatment. The study concluded that significant cardiotoxicity occurred in 6% of cases, mostly asymptomatic and reversible, and suggests that monitoring LVEF could potentially be reduced after 6-9 months if no early signs of cardiotoxicity are detected. This provides valuable insights into the cardiac safety of these treatments in real-world settings.

Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ.

Background: In melanomas, mutations in the BRAF gene are common and their occurrence represents an early oncogenic event. Our goal was to determine and compare the frequency of BRAF gene mutations in dysplastic nevi (ND) and melanomas in situ (MIS), as well as whether there is a correlation between the presence of BRAF gene mutations and various anamnestic, clinical, and histopathologic variables. Methods: A total of 175 patients-106 with ND, 41 with MIS, and 28 with lentigo maligna (LM) were included in the study. DNA was extracted from tissue samples and analyzed using the competitive allele-specific TaqMan chain reaction by polymerase in real time to detect the presence of BRAF V600E and V600K mutations. The data were compared with anamnestic, clinical, and histopathological data. Results: There is a statistically significant correlation between the presence of BRAF mutation and the diagnosis of melanoma in situ (χ2 test, χ2 = 29.17, p < 0.0001). Patients with LM had a significantly lower incidence of BRAF mutations compared to patients with ND and MIS. There was a significant correlation between the presence of a BRAF mutation and tumor localization, as well as the age of the patient, but no statistically significant correlation between the presence of a BRAF mutation and sex, tumor size, or previous melanoma diagnosis. Conclusions: BRAF mutations in ND are essentially required; however, they are an insufficient oncogenic trigger for the development of melanoma. This research contributes to a better understanding of the etiopathogenesis of melanoma and the role of ND as possible precursor lesions.

Intriguing Insights From 100 Consecutive Colorectal Cancer Cases in Mid-Kerala: Sparse BRAF Gene Mutations and Mismatch Repair Deficiency (MMR-D).

Colorectal cancer (CRC) is among the most prevalent types of cancer globally. It is well established that the development of CRC primarily results from the sequential activation of oncogenes and the simultaneous inactivation of tumor suppressor genes. It has also been noted that after the initial oncogenic mutation, many subpopulations with different mutational profiles are created, causing heterogeneity among the tumors. This retrospective study analyzed 100 patients diagnosed with CRCthrough colectomy over an eighteen-month period at a tertiary referral center in mid-Kerala, India. Pathology records and histological slides were reviewed by two pathologists, and clinicopathological data were collected from pathology reports. Immunohistochemical analysis for BRAF mutation and possible microsatellite instability (MSI) (by mismatch repair (MMR) protein study) was conducted on tumor tissue blocks sent to an external center due to the lack of an automated platform at the hospital. The study utilized Roche's Benchmark XT platform for BRAF analysis and assessed MMR protein expression using antibodies for MLH1, MSH2, MSH6, and PMS2. The mean age of patients was 58.36 years, with a male predominance (58.0%). Most tumors were classified as T3 (71.0%, n-71) and T2/T4a (14.0% each, n-14), while nodal involvement included N0 (35.0%, n-35), N1 (26.0%,n-26), N2 (19.0%, n-19), and NX (20.0%, n-20). Histological examination revealed predominantly well-differentiated tumors (78.0%, n-78), with lymphatic invasion noted in 41.0% (n-41) and vascular invasion in 5.0% (n-5) of cases. Left-sided tumors predominated (33.0%, n-33), followed by rectal carcinoma (37.0%, n-37), and right-sided colon cancers (30.0%, n-30). Genetic profiling showed sparse BRAF mutations (1.0%, n-1) and MSI (1.0%, n-1), with some cases exhibiting loss of MMR proteins (MLH1, PMS2, MSH2, and MSH6) by immunohistochemistry (IHC). The study highlights the rarity of BRAF mutations in this cohort and emphasizes the diverse pathological and molecular characteristics observed. The discussion focuses on the implications of these findings, suggesting that CRC in this population exhibits unique clinicopathological features potentially influenced by factors beyond genetic mutations. Further multicentric studies are warranted to comprehensively explore these factors and refine risk stratification and treatment strategies for CRC patients in similar demographics.

Clinicopathological significance and prognostic analysis of p21 and EGFR in colorectal cancer: a retrospective analysis on 12 319 cases in China

AimsColorectal cancer (CRC) is the third most common malignancy worldwide. Accurate pathological diagnosis and predictive abilities for treatment response and prognosis are crucial for patients with CRC. This study aims...

Development of custom amplicon-based test systems for detecting KRAS, NRAS, and BRAF gene mutations in colorectal cancer using next-generation sequencing (NGS).

e15061 Background: Polymerase chain reaction (PCR)-based mutation diagnostics in colorectal cancer is widely available. However, PCR has limitations in identifying rare mutations and precise mutation coordinates. Targeted sequencing, though promising, often incurs high costs and longer research durations, demanding higher DNA sample quality. Our study demonstrates the benefits of using a customized amplicon test system for diagnosing somatic mutations in tumor tissue, including detecting rare mutations not captured by PCR, requiring less input biomaterial, and similar research costs. Methods: A targeted NGS assay system was developed using region-specific primers for KRAS (exons 2, 3, and 4), NRAS (exons 2, 3, and 4), and BRAF (exon 15). Library preparation involved a three-step PCR process: Target PCR incorporating Illumina overhang adapter sequences, Index PCR introducing multiplexing indices and sequencing adapters, and PCR cleanup using magnetic beads. Sequencing utilized MiSeq v2 reagents with paired 300‐bp reads, generating high-quality, full-length reads. Validation included re-testing known samples. Tumor cell percentages in FFPE specimens were determined, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Data analysis utilized proprietary software. Results: Out of 1234 samples, 1133 were tested, with mutations detected in 685 samples (60.45%). KRAS mutations were found in 548 (48.36%), NRAS mutations in 40 (3.5%), and BRAF mutations in 88 (7.7%) samples. Double mutations were observed in 9 samples (0.79%). Data on rare and double mutations are presented in the table. Conclusions: The developed test system accurately identifies essential mutations in colorectal cancer, including rare and potentially clinically significant variants. The required DNA amount ranged from 10 to 30 ng in 5 μl, nearly ten times less than well-known commercial PCR test systems like COBAS (Roche) and therascreen (QIAGEN). The cost of the study is comparable to PCR with a minimum study duration of 4 days. [Table: see text]

Clinical and epidemiological profile of patients with atypical melanomas treated with immunotherapy.

e21532 Background: Atypical melanomas, such as acral lentiginous, uveal, mucosal, spitzoid, desmoplastic, meningeal, and primary occult site, represent less than 5% of melanoma cases. Due to their different biological tumor behavior from cutaneous melanomas, their treatment is still challenging. Currently, checkpoint inhibitors are the main treatment based on extrapolations or data from small surveys. Methods: This study aims to describe the epidemiological profile, clinical and pathological characteristics, and treatment of patients with atypical melanomas treated with immunotherapy. It is an observational, descriptive, retrospective study that analyzed 72 medical records of patients with atypical melanomas, ECOG 0-1, &gt; 18 years old, treated with immunotherapy (neo, adjuvant, and palliative lines), between January 2015 and December 2023 at the A.C. Camargo Cancer Center, São Paulo, Brazil. Results: We gathered 72 patients, 61% male and 39% female, with an average age of 58.89 (±13.63) years; 57% were white, 15% African-descendant (black and brown), 8% Asian, and 20% not specified. Regarding primary site, 26% were acral, 24% mucosal, 24% occult primary site, 15% uveal, 8% desmoplastic, 1% amelanocytic, 1% spitzoid, and 1% cauda equina meningeal. Regarding staging, (3%) were stage I, (15%) stage II, (25%) stage III, (47%) stage IV, and (10%) unknown. LDH: 372.5 (±360) mg/dL. Metastases: lymph node 29%; hepatic 23%; pulmonary 21%; bone 18%; central nervous system 15%; peritoneal carcinomatosis 5%; stomach 3%; cauda equina 3%; adrenal 1%; in transit 1%; intestine 1%; bone marrow 1%; parotid 1%. BRAF mutations were present in 23% of cases (variants V660E, V600K, V660R, and G469R); NRAS 6% (codons 13 and 61); KIT 1% (exon 11) and NTRAK 1% (S701F). In relation to treatment, 5 received neoadjuvant anti-pd1 + anti-ctla4 combination, 13 adjuvant with anti-pd1+/- anti-ctla4. About 50 patients received systemic treatment for metastatic disease anti-pd1+/- anti-ctla4. Overall survival: 26 (±14.8) months. Conclusions: Atypical melanoma primary occult site is the most prevalent, with the majority diagnosed at stage IV, with high LDH levels and lymph nodes as the main sites of metastases. Twenty-three percent had a BRAF gene mutation. The majority were treated palliatively with first-line immunotherapy, with median survivals of 14.8 months.

Liquid biopsy multiomics enable clinical triage for early colorectal cancer diagnosis.

e15599 Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United State. Despite advancements in screening, diagnosis, and treatment, the disease remains a formidable public health issue. In 2023, approximately 153020 individuals were diagnosed with CRC and 52550 will die from the disease. In order to meet early diagnostic, we developed this multiomics assay to enable clinical triage for liquid biopsy screening. Methods: Xenonucleic acid (XNAs) are artificial genetic polymers that retain the Watson-Crick base-pairing capability and exhibit high chemical and biological stability. They can be employed as molecular clamps in RT-qPCR due to the stronger hybridizing/binding capability seen in XNA/DNA duplexes than DNA/DNA duplexes. We are developing an XNA-based multiplex RT-qPCR assay for APC, BRAF, CTNNB1, KRAS, NRAS, PIK3CA, SMAD4 and TP53 gene mutation panel and CRC 9 genes methylation panel combined with multiplex protein panel, called ColoscapeTM Plus. We enrolled in FIT positive subjects of both genders, aged 50-74, attending the CRC screening program, we tested 152 plasma samples, 75 health, 50 AA and 27 early CRC with this multiomics assay. Results: The assay results showed that its sensitivity was about 44% (95% CI: 0.302- 0.587) and specificity about 80% (95%CI: 0.689- 0.880) for advanced adenoma (AA). For CRC stage I and II, its sensitivity is about 63% and 78% with 80% specificity separately. Conclusions: The primary data has shown that this low cost and rapid turnaround time multiomics assay has a comparable sensitivity for early colorectal cancer diagnostic.

Incidence of common gene mutations in early-onset colorectal cancer and the association with cancer survival: a meta-analysis

Objective: The incidence of early-onset colorectal cancer (EOCRC) is increasing globally; however, the molecular characteristics and prognosis of sporadic EOCRC are unclear. In this systematic review and meta-analysis, we aimed to investigate the incidence of gene mutations and their association with cancer survival in sporadic EOCRC, focusing on six common gene mutations (TP53, BRAF, KRAS, NRAS, PTEN, and APC). Methods: Ovid Embase and Ovid Medline electronic databases were searched for studies involving patients with sporadic EOCRC (i.e., diagnosed with colorectal cancer before the age of 50 years and with no evidence of hereditary syndromes predisposing to colorectal cancer). The included articles were evaluated using quality assessment tools. Meta-analysis was performed using random-effects and fixed-effects models. Cochran's Q statistic and the I2 index were used to assess heterogeneity. The incidence of the six common gene mutations listed above in sporadic EOCRC and their association with cancer survival were evaluated. Results: (1) Incidence of specific gene mutations in sporadic EOCRC. A total of 34 articles were included in this meta-analysis. The incidence of APC gene mutation was 36% (from 13 articles, 95%CI: 19%-55%, P=0.043); of KRAS gene mutation 30% (from 26 articles, 95%CI: 24%-35%, P=0.190); of BRAF gene mutation 7% (from 18 articles, 95%CI: 5%-11%, P=0.422); of NRAS gene mutation 4% (from five articles, 95%CI: 3%-5%, P=0.586); of PTEN gene mutation 6% (from six articles, 95%CI: 4%-10%, P=0.968); and of TP53 gene mutation 59% (from 13 articles, 95%CI: 49%-68%, P=0.164). (2) Association between gene mutations and survival in sporadic EOCRC. A total of six articles were included in this meta-analysis. Compared with wild-type BRAF, mutant BRAF was significantly associated with increased overall mortality risk in patients with EOCRC (pooled HR=2.85, 95%CI: 1.45-5.60, P=0.002). Subgroup analysis showed that the incidence of BRAF gene mutation was higher in Eastern than in Western countries, whereas the incidence of TP53, KRAS, NRAS, and APC gene mutations was lower. There was no significant difference in the incidence of PTEN gene mutation between different regions. Conclusion: Compared with colorectal cancer occurring in the general population, the incidence of APC and KRAS mutations is lower in EOCRC, whereas the incidence of TP53 mutation remains consistent. BRAF mutation is associated with increased overall mortality risk in patients with EOCRC.

Value of Multimodal Ultrasound Combined with BRAF Gene in Evaluating Cervical Lymph Node Metastasis of Papillary Thyroid Microcarcinoma

ObjectiveThe aim of the work described here was to explore the predictive value of multimodal ultrasound combined with the BRAF gene in cervical lymph node metastasis (CLNM) of papillary thyroid microcarcinoma (PTMC). MethodsOne hundred six patients (114 lesions) with PTMC confirmed by surgery and pathology at Yantai Yuhuangding Hospital from July 2021 to August 2022 were analyzed retrospectively. Routine ultrasound, contrast-enhanced ultrasound, shear wave elastography examination and BRAF V600E gene detection were performed before surgery. Patients were divided into two groups on the basis of post-operative pathology: non-metastasis group and metastasis group. Univariate and multivariate analyses were used to analyze the risk factors of cervical lymph node metastasis in PTMC. ResultsUnivariate analysis revealed that there were significant differences in gender, high echo in lesions, enhancement level, peak intensity (PI) and average modulus of elasticity (Eavg) between the two groups (p < 0.05), but there was no significant difference in BRAF gene mutation (p = 0.855). Multivariate analysis revealed that male gender, microcalcification and hyper- or iso-enhancing parametric increased the risk of CLNM in PTMC (p < 0.05), and that sensitivity (92.3%) and accuracy (73.9%) were higher for combined diagnosis than for single diagnosis; the differences were statistically significant (p < 0.05). ConclusionMale gender, microcalcification and hyper- or iso-enhancing parametrics of CEUS are independent risk factors for CLNM in PTMC patients. Combined diagnosis is more effective.