Presence Of BRAFV600E Research Articles

Association Between Gross Features and Coexistence of BRAFV600E and TERT Promoter Mutations in Papillary Thyroid Carcinomas: A Combined Analysis Incorporating Clinicopathologic Features.

Background: The coexistence of v-Raf murine sarcoma viral oncogene homolog B1 (BRAFV600E) and telomere reverse transcriptase promoter (TERT-p) mutations is considerably associated with aggressiveness and poor prognosis in papillary thyroid carcinoma (PTC). However, the association between gross findings and genetic alterations in PTC remains unknown. We aimed to investigate the association between clinicopathologic features, including macroscopic features, and the coexistent BRAFV600E and TERT-p mutations in patients with PTC. Methods: We retrospectively analyzed 375 cases of PTC surgically resected between January 2018 and October 2023 at a single institution, based on the presence of BRAFV600E and TERT-p double mutation. Clinicopathologic features, including gross features on the cut surface of tumors, were evaluated. Subsequently, the association between clinicopathologic features and mutation status was statistically examined. Cox proportional hazard models were used to analyze the impact of molecular pathological features on disease-free survival (DFS). Results: The BRAFV600E and TERT-p double mutation was identified in 78 (20.8%) patients among the PTC cases and was significantly correlated with shorter DFS. Multivariable analysis revealed that factors such as relatively older age (≥55 years) (odds ratio [OR] = 12.083, 95% confidence interval [CI] 4.498-32.456), larger tumor size (>2.0 cm) (OR = 2.722, CI 1.104-6.712), lobulated tumor margins (OR = 16.114, CI 3.155-82.296), papillary excrescences on the cut surface (OR = 17.573, CI 3.462-89.201), solid-cut surface (OR = 4.012, CI 1.084-14.849), minimal extrathyroidal extension (ETE) (OR = 4.156, CI 1.209-14.282), gross ETE (OR = 6.517, CI 1.734-24.490), and Ki-67 labeling index (LI) (≥5%, OR = 12.145, CI 4.354-33.877) were significantly associated with the double mutation. Conclusions: The BRAFV600E and TERT-p double mutation in PTC was significantly associated with relatively old age, larger tumor size, lobulated configuration in tumor margin, papillary excrescences on the cut surface, solid-cut surface, ETE, and high Ki-67 LI. These features are suggestive of the presence of the double mutation and should be analyzed at the molecular level in patients with PTC.

7962 Transformation of Poorly Differentiated Papillary Thyroid Carcinoma to Anaplastic Thyroid Carcinoma, an Atypical Course!

Abstract Disclosure: T. Altaweel: None. S. Makadsi: None. M. Hussein: None. J. Samantray: None. Introduction: Anaplastic thyroid cancers are undifferentiated, rare and aggressive. The transformation of papillary thyroid cancer to anaplastic thyroid cancer is documented in literature, however, rare. In this report, we present a case of Anaplastic thyroid carcinoma likely arising from a co-existing poorly differentiated Papillary carcinoma. Case Presentation: A 74-year-old male with past medical history of tobacco smoking who presented with the onset of voice hoarseness for 6 months. He has also noticed a left neck mass that is in the mid supraclavicular region. A Neck CT neck was performed at the time of presentation and was significant for a 4.6x3.4x5.9 cm left thyroid mass with displacement of the trachea. FNA biopsy showed papillary carcinoma with focal features of Tall Cell Variant. Poorly differentiated components with fragments of stroma and muscle were seen and suggested invasion. These findings raised the possibility of anaplastic thyroid carcinoma in the setting of papillary carcinoma and poorly differentiated carcinoma. BRAF V600E pathogenic variant mutation along with TERT mutation were found. Patient underwent direct laryngoscopy, bronchoscopy with biopsies, and tracheostomy with surgical pathology confirming papillary thyroid cancer with muscles invasion. Combination dabrafenib and trametinib were started. No surgical resection of thyroid tissue was planned. A follow up CT showed a significant decrease in the low-density soft tissue involving the medial thyroid gland bilaterally, consistent with interim positive response to therapy. Three months later, CT neck showed near complete resolution of low-density soft tissue involving the medial thyroid and parathyroid glands and subglottic trachea consistent with further interim positive response to therapy. Six months post initiation of therapy, CT Neck showed focal low density in the mid and inferior right thyroid gland which may represent residual and/or recurrent thyroid carcinoma. That density was further demonstrated on a repeat CT neck as a 1.8 cm hypodense nodule in the inferior right thyroid gland and concerning for recurrent thyroid tumor with possible infiltration of the trachea. Conclusion: ATC prognosis is affected by many factors such as metastasis, age, treatment strategies, socioeconomic status and the presence of BRAF V600E and TERT mutations. Diagnosis can be challenging. Our patient presented months after his symptoms started and this is an atypical course of Anaplastic Thyroid Carcinoma as it usually progresses aggressively and rapidly. Given the pathology reports and the more slow presentation of this patient’s carcinoma, it can be suggested that this is a case of papillary carcinoma with a later transformation to ATC. In these cases, surgical resection Presentation: 6/2/2024

BRAF-V600E mutations in plasma and peripheral blood mononuclear cells correlate with prognosis of pediatric Langerhans cell histiocytosis treated with first-line therapy.

The clinical relevance of BRAF-V600E alleles in peripheral blood mononuclear cells (PBMCs) and the prognostic impact of the mutants in cell-free (cf) and PBMC DNAs of Langerhans cell histiocytosis (LCH) have not been fully clarified in pediatric LCH. We retrospectively determined the levels of BRAF-V600E mutation in paired plasma and PBMC samples at the time of diagnosis of LCH. Subsequently, we performed a separate or combined analysis of the clinical and prognostic impact of the mutants. We assessed BRAF-V600E mutation in peripheral blood from 94 patients of childhood LCH. Our data showed that cfBRAF-V600E was related to young age, multiple-system (MS) disease, involvements of organs with high risk, increased risk of relapse, and worse progression-free survival (PFS) of patients. We also observed that the presence of BRAF-V600E in PBMCs at baseline was significantly associated with MS LCH with risk organ involvement, younger age, and disease progression or relapse. The coexisting of plasma(+)/PBMC(+) identified 36.2% of the patients with the worst outcome, and the hazard ratio was more significant than either of the two alone or neither, indicating that combined analysis of the mutation in plasma and PBMCs was more accurate to predict relapse than evaluation of either one. Concurrent assessment of BRAF-V600E mutation in plasma and PBMCs significantly impacted the prognosis of children with LCH. Further prospective studies with larger cohorts need to validate the results of this study.

Predictors of receiving targeted treatment among eligible patients with metastatic colorectal cancer.

e15594 Background: Colorectal cancer is among the most frequently diagnosed cancers and ranks as the third leading cause of cancer-related mortality globally with increasing prevalence among younger patients and high mortality. While targeted therapies have revolutionized treatment and tripled the median survival over the past two decades, many eligible patients fail to receive them. This study sought to identify factors associated with receiving targeted treatment among eligible patients. Methods: We identified a cohort of patients with metastatic colorectal cancer diagnosed between 2007 and 2023 (n = 521,074) in Optum’s clinicogenomic database. Eligible patients were identified based on clinical and treatment history, broad-panel next-gen sequencing (NGS) results and the drug indications for targeted treatments available at time of diagnosis (n = 8,533), including MSI/MMR status, RAS WT status, presence of BRAF V600E, and other biomarkers. In total, we considered tucatinib, encorafenib, pembrolizumab, nivolumab, ipilimumab, panitumumab, cetuximab, and regorafenib. Most patients did not receive targeted treatment; we applied a synthetic minority oversampling technique to correct this class imbalance. We assessed how factors including sex, race, and receipt of genetic counseling services influenced the likelihood of receiving targeted treatment using logistic regression. Results: In the fully adjusted multivariable logistic regression analysis, all variables were significant with p < 0.05. Notably, patients receiving genetic counseling had 68% greater odds of receiving targeted treatment (OR, 1.68 [95% CI, 1.20-2.36]). African American (OR, 0.37 [95% CI, 0.27-0.50]) and Asian patients (OR, 0.10 [95% CI, 0.04-0.24]) had lower odds of receiving targeted treatment compared to Whites. Similarly, patients treated in community settings had 86% lower odds compared to patients in academic settings (OR, 0.14, [95% CI, 0.11-0.18]) as did male patients (OR, 0.39, [95% CI, 0.33-0.44]). Conclusions: This study underscores the challenges in receiving targeted treatments despite clinical eligibility. Our findings highlight the significance of genetic counseling in facilitating access to targeted therapies, and the stark gap in uptake among minority patients. These results can inform strategies to increase access to these treatments. Future research will attempt to better understand the mechanisms underlying receipt of targeted treatment.

First Blood: Pre-Therapy BRAFV600E+ PBMC Identifies a Novel Clinical Risk Category for Patients with LCH

Purpose: Langerhans Cell Histiocytosis (LCH) is a myeloproliferative neoplasia driven by somatic mutations in the mitogen-activated protein kinases pathway (MAPK). LCH has historically been clinically staged as “high risk” (e.g. liver, spleen and/or bone marrow involvement, HR) or “low risk” (LR) due relative to risk of death in earlier trials. However, clinical staging is complicated by different sensitivities of imaging and, more recently, molecular analyses. We previously identified that BRAFV600E allele in peripheral blood mononuclear cells (PBMC) in patients with HR LCH, which supported a model of LCH arising from hematopoietic myeloid precursors. To determine the clinical significance of somatic lesion BRAFV600E mutations as well as ability to detect pre-therapy BRAFV600E+ PBMC at diagnosis, we analyzed an extended cohort of LCH patients with longitudinal clinical evaluations. Methods: Genomic DNA was isolated from 366 lesion biopsies and the PBMCs of 294 patients with active LCH before initial therapy. The percentage of PBMC with the BRAFV600E allele in patients was determined by quantitative polymerase chain reaction. Lesion genotype and ability to detect pre-therapy BRAFV600E in PBMC were correlated with clinical features including i) clinical risk status, ii) extent of disease (single lesion vs multifocal; single system vs multisystem), iii) response to therapy, v) and time to relapse were analyzed using a chi-square test. Fluorescence-activated cell sorting (FACS), DNA amplification and subsequent qPCR was performed on 15 pretherapy BRAFV600E+ blood samples to explore the relationship between different myeloid lineages (CD3, CD19, CD56, CD14, CD16, CD11c) and extent of LCH disease. Survival proportions were calculated using the Kaplan-Meier (product limit) method and a log-rank (Mantel-Cox) test was used to calculate the significance of the difference between survival curves. Results: Median age was 3.01 years (range 3.6 days to 21.5 years), 58.5% of the patients were male (41.5% female), and 15.4% had HR disease (84.6% LR). Among patients with LR disease, 33.0% had multisystem, 23.1% had multi-focal single system, and 44.0% had single lesions. Lesion genotypes were 51.9% BRAFV600E and 49.1% not BRAFV600E. BRAFV600E in LCH lesions was associated with lower age at diagnosis and higher prevalence of HR disease. However, after a median follow-up time of 1.6 years (range 1 month to 8.5 years) there was no difference in EFS between BRAFV600E and alternative mutations (p=0.35). Notably, for the entire cohort, presence of BRAFV600E in diagnostic PBMC was highly associated with younger patients, clinical HR disease and risk of developing LCH-associated neurodegeneration (LCH-ND). For patients with BRAFV600E+ lesions, 32.5% had detectable BRAF V600E in pre-therapy PBMC: 100% of HR, 52.6% of multisystem LR, 18.4% of multi-focal single system LR and 0% of single lesion. Further, after a median follow-up time of 2.0 years (range 1 month to 9,1 years) clinical LR PBMC BRAFV600E+ represented a group at higher risk of treatment failure compared to BRAFV600E-neg LR (p=0.01). BRAFV600E alleles were present in PBMC at diagnosis in all of the sorted myeloid populations, with consistent enrichment in myeloid dendritic cells and monocytes. There was no significant correlation between BRAFV600E allele burden (percentage of BRAFV600E+ PBMC) and clinical risk, and no significant differences in the distribution of allele burden within lineages. Conclusion: Ability to detect pretherapy BRAFV600E+ represents an important clinical biomarker for patients with LCH. Results from this study support the potential for pre-therapy PBMC BRAFV600E to predict clinical risks, inform pathogenic mechanisms in LCH, and identify LR PBMC BRAFV600E+ as a novel clinical risk group. We hypothesize that persistence of hematopoietic clones and circulating myeloid precursors may underlie the increased risks of treatment failure and development of LCH-ND in patients previously presumed to have “low risk” disease.

Abstract 928: Correlation among actionable gene mutations, microsatellite instability and tumor mutational burden in advanced colorectal cancer and association with survival

Abstract Introduction: Targeted therapies and immunotherapies have changed the treatment strategy for advanced colorectal cancer. Characterizing associations of actionable gene mutations with microsatellite instability (MSI) and tumor mutational burden (TMB) could suggest mechanistically intriguing combinations of targeted and immunotherapies. Methods: Publicly available genetic and overall survival (OS) data of patients with colorectal cancer were analyzed. (N=3,548, MSK-MET through cBioPortal for cancer genomics) We investigated the correlations among actionable gene mutations (BRAF V600E, KRAS G12C, HER2 amplification, NTRK fusion, and RET fusion), MSI and TMB (>20/Mb). The association of actionable mutations, MSI and TMB patterns with OS was analyzed. Results: BRAF V600E, KRAS G12C, HER2 amplification, NTRK fusion, RET fusion, MSI and TMB >20 was observed in 6.9, 3.1, 2.8, 0.3, 0.2, 9.2 and 9.9%, respectively. BRAF V600E showed mutual exclusivity toward KRAS G12C and HER2 amplification. MSI and TMB > 20 were associated with the presence of BRAF V600E, NTRK fusion, and RET fusion and the absence of KRAS G12C and HER2 amplification. MSI and TMB > 20 were highly correlated. (Table) Among patients with MSI (N=316), BRAF V600E was associated with shorter OS. (Hazard ratio (HR) 2.22, 95% confidence interval (CI); 1.12 - 4.41) In the patients without MSI, TMB >20 showed significantly longer OS, compared with TMB ≤20. (HR 0.26, 95% CI; 0.085 - 0.82) Association of NTRK or RET fusion with survival could not be reliably assessed due to small sample size (n < 10 for both). Conclusion: There is significant correlation among targetable genetic mutations, MSI and TMB in colorectal cancer, and biomarker profiles are associated with differences in OS. Future studies should evaluate whether combinations of targeted therapies and immunotherapy have better efficacy than monotherapy approaches in selected patients. Correlation among actionable gene mutations and immunomic signatures (OR and 95 % CI) KRAS G12C HER2 amp NTRK fusion RET fusion MSI TMB >20 BRAF V600E 0 (0 - 0.44) * 0 (0 - 0.49) * 0 (0 - 6.79) 0 (0 - 9.31) 10.0 (7.39 - 13.5) * 8.76 (6.52 - 11.7) * KRAS G12C 0.31 (0.01 - 1.78) 0 (0 - 16.0) 0 (0 - 21.9) 0.18 (0.02 - 0.67) * 0.16 (0.02 - 0.61) * HER2 amp 0 (0 - 17.5) 0 (0 - 23.9) 0.10 (0.00 - 0.57) * 0.09 (0.00 - 0.51) * NTRK fusion 0 (0 - 315) 20.0 (4.25 - 124) * 18.4 (3.91 - 115) * RET fusion 24.9 (4.06 - 261) * 23.0 (3.74 - 241) * MSI 1.52 x 104 (4.20 x 103 - 4.50 x 1015) * Citation Format: Hirotaka Miyashita, Gabriel Brooks, Razelle Kurzrock, Shumei Kato. Correlation among actionable gene mutations, microsatellite instability and tumor mutational burden in advanced colorectal cancer and association with survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 928.

Epidemiological and molecular evaluation of BRAF, KRAS, NRAS genes and MSI in the development of colorectal cancer

Background and ObjectivesKRAS, NRAS, BRAF mutations and microsatellite instability (MSI) can be associated with Colorectal Cancer (CRC) development. Material and MethodsWe evaluated 828 medical records of CRC patients from a school hospital from January/2016 to December/2020. Variables such as age, gender, ethnicity, literacy level, smoking, alcoholism, primary anatomical site, tumor staging, presence of BRAFV600E, KRAS, NRAS mutations and MSI , survival and metastasis were identified. The statistical analyses were performed (p < 0.05 was considered significant). ResultsThere was a predominance of males (51.93%), whites (90.70%), low education (72.34%), smokers (73.79%), and non–alcoholics (79.10%). Rectum was the most affected site (42.14%), advanced tumor stage was most prevalent (62.07%), and metastasis occurred in (64.61%). Of the enrolled patients; 204 were investigated for BRAF mutation and detected in (2.94%); 216 for KRAS gene and detected in (26.08%); 210 for NRAS gene, and detected in (25.36%); 370 for MSI and detected in (44.68%). A significant association of CRC with NRAS mutation and alcohol habit (p = 0.043) was observed. The presence of MSI was associated with primary site proximal colon (p < 0.000), distal colon (p = 0.001) and rectum (p = 0.010). ConclusionPatients with CRC are male, over 64 years old, white, with low education, smokers and non-alcoholics. The most affected primary site is rectum in advanced stage with metastasis. CRC is associated with NRAS mutation and alcohol habit, there is increased risk for primary site of proximal colon and MSI; decreased risk for distal colon and rectum in the presence of MSI.

Tumor mutation burden-assisted risk stratification for papillary thyroid cancer.

Although papillary thyroid cancer (PTC) has a low mortality rate, the rate of recurrence remains relatively high. This study aims to develop a molecular signature to predict the recurrence of PTC. A total of 333 PTC patients' data from The Cancer Genome Atlas (TCGA) were included. We calculated tumor mutation burden (TMB) and analyzed the mutation status of BRAF and TERT promoter. Tumor recurrence occurred in 17 of 263 cases in TMB-L patients versus 14 of 70 cases in TMB-H patients (hazard ratio [HR], 3.55; 95% confidence interval [CI], 1.75-7.21; P &lt; 0.001). The HR for recurrence in TMB-H patients remained significant after adjustment for classical clinicopathologic factors (patient age, gender, extrathyroidal extension and lymph node metastasis). These clinical factors had no effect on recurrence rate in TMB-L patients, but had a strong adverse effect on the prognosis of TMB-H patients. Compared with TMB-L patients lacking mutation, the HR (95% CI) of recurrence for TMB-H patients with coexisting BRAF V600E and/or TERT C228/250 T mutations was 6.68 (2.41-18.57), which remained significant after adjustment for clinicopathological factors. The mutation status of BRAF V600E and TERT C228/250 T had little effect on PTC recurrence in TMB-L patients. Either of the mutation was associated with high recurrence rate in TMB-H patients. The presence of BRAF V600E and/or TERT promoter mutations denotes a high risk of recurrence in TMB-H patients. This represents a powerful molecular prognostic genotype that can help predict patients with the highest risk of recurrence.

Abstract 5245: Characterization of sub-clonal RAS/BRAF alterations in an anti-EGFR treated advanced CRC cohort using a liquid biopsy-based real world clinical genomics database

Abstract Background: Anti-EGFR monoclonal antibodies are treatment options for RAS and BRAF V600 mutation-negative CRC patients. However, the literature suggests that CRC patients with sub-clonal RAS and BRAF mutations may still benefit from anti-EGFR therapies. The Guardant INFORM™ real-world clinical-genomic database was utilized to assess the impact of sub-clonal RAS and BRAF alterations detected in circulating tumor DNA (ctDNA) by the Guardant360® test on the clinical outcome of CRC patients treated with anti-EGFR therapy. Methods: Patients were selected from the Guardant INFORM™ database using the following inclusion criteria: diagnosed with advanced CRC in the US; treated with anti-EGFR therapies starting within 90 days after a Guardant360 test result; and the presence of BRAF V600E and/or canonical KRAS/NRAS mutations (codons 12, 13, 59, 61, 117, 146). Time to next treatment (TTNT) and overall survival (OS) were compared for various RAS/BRAF mutation clonality cutoffs using the Cox proportional hazards model. Results: Of the 13,798 CRC patients in the Guardant INFORM database, 91% had detectable ctDNA, 913 received a Guardant360® test before anti-EGFR therapy, and 446 (48.8%) initiated anti-EGFR therapy within 90 days after the test. Among this cohort, 11% (n = 50) had a BRAF V600E mutation and 9% (n = 40) had a RAS mutation. The median RAS/BRAF clonality was 0.84 (IQR = 0.57, 1.00). The associated clinical outcomes for patients with sub-clonal and clonal RAS/BRAF mutations using clonality cutoffs from 0.3 to 0.8 are reported (Table). Conclusion: Using a liquid biopsy-based clinical-genomic dataset, we demonstrate that patients harboring sub-clonal RAS or BRAF mutations benefit from anti-EGFR therapy to a degree similar to wild-type patients. Given the limited patient numbers, this finding warrants additional investigation into the utility of ant-EGFR therapies as a treatment option for this subgroup of CRC patients. RAS/BRAF Mutation Clonality* Cutoff Sub-clonal** Clonal** HR (95% CI) P value HR (95% CI) P value TTNT 0.3 0.96 (0.42, 2.18) 0.913 1.72 (1.15, 2.59) 0.009 0.4 1.03 (0.48, 2.21) 0.946 1.72 (1.14, 2.60) 0.010 0.5 0.97 (0.45, 2.08) 0.931 1.77 (1.17, 2.66) 0.007 0.6 1.04 (0.51, 2.13) 0.922 1.76 (1.16, 2.66) 0.008 0.7 1.03 (0.55, 1.94) 0.919 1.90 (1.23, 2.93) 0.004 0.8 1.30 (0.75, 2.24) 0.350 1.72 (1.08, 2.74) 0.022 OS 0.3 1.72 (0.84, 3.54) 0.142 2.47 (1.61, 3.79) &amp;lt;0.001 0.4 1.85 (0.93, 3.66) 0.079 2.42 (1.57, 3.74) &amp;lt;0.001 0.5 1.79 (0.90, 3.54) 0.097 2.46 (1.59, 3.80) &amp;lt;0.001 0.6 1.92 (1.00, 3.69) 0.050 2.40 (1.55, 3.73) &amp;lt;0.001 0.7 1.83 (1.03, 3.26) 0.041 2.60 (1.63, 4.14) &amp;lt;0.001 0.8 2.16 (1.29, 3.61) 0.003 2.33 (1.40, 3.86) 0.001 *Clonality = (RAS or BRAF MAF)/(maximum MAF). **Reference group is patients without RAS/BRAF mutations. Citation Format: Khalid Abubaker, Danyi Wang, Zheng Feng, Christopher Stroh, Jiemin Liao, Gordon Otto, Juergen Scheuenpflug. Characterization of sub-clonal RAS/BRAF alterations in an anti-EGFR treated advanced CRC cohort using a liquid biopsy-based real world clinical genomics database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5245.

Identification of BRAF V600E mutation in odontogenic tumors by high-performance MALDI-TOF analysis

Odontogenic tumors are rare lesions with unknown etiopathogenesis. Most of them are benign, but local aggressiveness, infiltrative potential, and high recurrence rate characterize some entities. The MAP-kinase pathway activation can represent a primary critical event in odontogenic tumorigenesis. Especially, the BRAF V600E mutation has been involved in 80–90% of ameloblastic lesions, offering a biological rationale for developing new targeted therapies. The study aims to evaluate the BRAF V600E mutation in odontogenic lesions, comparing three different detection methods and focusing on the Sequenom MassARRAY System. 81 surgical samples of odontogenic lesions were subjected to immunohistochemical analysis, Sanger Sequencing, and Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (Sequenom). The BRAF V600E mutation was revealed only in ameloblastoma samples. Moreover, the presence of BRAF V600E was significantly associated with the mandibular site (ρ = 0.627; P value <0.001) and the unicystic histotype (ρ = 0.299, P value <0.001). However, any significant difference of 10-years disease-free survival time was not revealed. Finally, Sequenom showed to be a 100% sensitive and 98.1% specific, suggesting its high-performance diagnostic accuracy. These results suggest the MAP-kinase pathway could contribute to ameloblastic tumorigenesis. Moreover, they could indicate the anatomical specificity of the driving mutations of mandibular ameloblastomas, providing a biological rational for developing new targeted therapies. Finally, the high diagnostic accuracy of Sequenom was confirmed.

Predictive value of CD138, CD68, CD163 expression in the pathogenesis of the development of thyroid cancer against the background of metabolic disorders

Objective — to investigate the expression of CD138, CD68, CD163, involved in the pathogenesis of papillary thyroid cancer (PTC) against the background of metabolic disorders. Materials and methods. The analysis was performed on 36 selected medical histories of patients who were operated for PTC in the Surgical Department of the Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues of the MoH of Ukraine. Patients’ aged ranged 18 to 76 years, the mean age was 41.4 ± 13.8 years; women prevailed among patients — 32. According to the body mass index (BMI) patients were divided into two groups: group 1 (n = 14) with normal weight (BMI &lt; 25 kg/m2), group 2 consisted of 22 overweight patients (BMI ≥ 25 kg/m2). All patients had autoimmune thyroiditis confirmed by laboratory and histological examination. Molecular genetic mutation BRAFV600E was detected in 20 (55.6 %). Immunohistochemical study of markers CD138, CD68, CD163 was performed according to the standard methods on histological material of tumors. Results. Among the studied immunohistochemical markers, CD68 was the most common — 72.2 % of cases, while CD138 and CD138 were found in 50 % and 55.6 %, respectively. In both studied groups, no statistically significant association was found between BMI and the presence of BRAFV600E (p &gt; 0.05). In the immunohistochemical study of markers CD138, CD68 and CD163 no significant relationship between their expression and BMI was found (p &gt; 0.05). Instead, we found a significant difference in the detection of the CD68 marker in patients of both groups (p &lt; 0.01), while CD138 and CD163 did not show significant difference. The aggressiveness of the tumor process occurred in 25 % of patients. Patients in group 1 (with normal BMI) did not have significant differences in the aggressiveness in contrast to patients of the group 2 (21.4 % and 27.3 %, respectively). Conclusions. No increase of the expression of immunohistochemical markers CD138, CD68, CD163, involved in the pathogenesis of PTC, was established in overweight patients; there was no significant correlation between BMI and the presence of BRAFV600E, as well as the aggressiveness of the tumor process.

Transcriptomic landscape of circulating mononuclear phagocytes in Langerhans cell histiocytosis at the single-cell level

AbstractLangerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm caused by aberrant activation of the mitogen-activated protein kinase (MAPK) pathway. Circulating myeloid cells from patients often carry disease-associated mutations and can be differentiated into langerinhigh LCH-like cells in vitro, but their detailed immune-phenotypic and molecular profiles are lacking and could shed key insights into disease biology. Here we recruited 217 pediatric LCH patients and took blood and tissue samples for BRAFV600E analysis. Immune-phenotyping of the circulating Lin−HLA-DR+ immune population in 49 of these patients revealed that decreased frequency of plasmacytoid dendritic cells was significantly linked to disease severity. By single-cell RNA sequencing of samples from 14 patients, we identified key changes in expression of RAS-MAPK-extracellular signal-regulated kinase (ERK) signaling-related genes and transcription factors in distinct members of the mononuclear phagocyte system in the presence of BRAFV600E. Moreover, treatment of patients with the BRAF inhibitor dabrafenib resulted in MAPK cascade inhibition, inflammation prevention, and regulation of cellular metabolism within mononuclear phagocytes. Finally, we also observed elevated expression of RAS-MAPK-ERK signaling-related genes in a CD207+CD1a+ cell subcluster in skin. Taken together, our data extend the molecular understanding of LCH biology at single-cell resolution, which might contribute to improvement of clinical diagnostics and therapeutics, and aid in the development of personalized medicine approaches.

BRAFV600E mutation is associated with aggressive features in papillary thyroid carcinomas\u2009\u2264\u20091.5\xa0cm

BackgroundWhile some studies suggest that the BRAF V600E mutation correlates with a high-risk phenotype in papillary thyroid microcarcinoma (PTMC), more evidence is necessary before this mutation can be used to help guide decision making in the management of small thyroid nodules. This study investigated whether BRAF V600E mutation is associated with aggressive features in PTMC (≤ 1 cm) and small PTC (1–1.5 cm).MethodsRetrospective chart review was performed on 121 patient cases. Patients who underwent thyroid surgery for PTMC (≤ 1 cm) or small PTC (1–1.5 cm) were included if molecular testing was done for BRAF V600E mutation. Two study groups were created based on tumour size: PTMC (n = 55) and small PTC (n = 66). The groups were analysed for the presence of a BRAF V600E mutation and aggressive features, including macroscopic extrathyroidal extension (ETE), lymph node metastasis (LNM), and high-risk histological features (tall cell, columnar cell, hobnail, solid/trabecular, and diffuse sclerosing). The Fischer exact test was used to calculate statistical significance.ResultsBRAF V600E mutations were detected in 43.6% of PTMC and 42.4% of small PTC. Of the mutated PTMC nodules, 54.1% demonstrated aggressive characteristics as compared to 19.4% of the non-mutated PTMCs (p = 0.010). Of the mutated small PTC tumours, 82.1% had aggressive features. In contrast, 28.9% of the non-mutated small PTCs showed aggressive features (p < 0.001).ConclusionsOur findings demonstrate an association between a BRAF V600E mutation and aggressive features in PTMC (≤ 1 cm) and small PTC (1–1.5 cm). Therefore, determining the molecular status of these thyroid nodules for the presence of BRAF V600E can help guide patient management.Graphical

INNV-06. SECOND GENERATION BRAF/MEK INHIBITION IN ANAPLASTIC PLEOMORPHIC XANTHROASTROCYTOMA

Abstract BACKGROUND Up to 65% of anaplastic pleomorphic xanthoastrocytomas (A-PXA) harbor the BRAFV600E oncogene. Trials and case series have demonstrated that BRAF-mutant gliomas, including A-PXAs, can be responsive to the first generation BRAF inhibitors dabrafenib and vemurafenib, with an ORR ranging from 26–43%. The second generation BRAF/MEK inhibitor combination encorafenib/binimetinib improves systemic outcome in melanoma patients, has better tolerability, and may have better intracranial activity compared to first generation drugs. METHOD We review the three patients with BRAFV600E A-PXA treated with encorafenib/binimetinib in our practice. RESULTS Two patients were diagnosed with A-PXA in 2014 and one in 2013. The presence of BRAFV600E was confirmed by molecular testing. All patients have received prior surgery, chemotherapy, and radiation and were at a recurrence when BRAF/MEK inhibition was initiated. One patient had CR after 2 months on treatment and remains in CR at 1-year. One patient, who is also receiving bevacizumab, has a PR, with continued clinical and imaging improvement at 6 months. Our third patient had a PR at 2 months and remains on therapy, currently 3 months post-initiation. All 3 patients report improvement in symptoms. The patient who has been on treatment for 1 year experienced rapid onset bilateral multifocal sub-retinal fluid collections followed by rapid resolution, without intervention, within days of starting treatment. This patient has also had mild decrease in EF over time. We will present continued follow up. CONCLUSIONS Encorafenib/binimetinib appears feasible, with 3 of out 3 patients with BRAFV600E A-PXA demonstrating radiographic response; 2 of these patients have durable responses. We stress the importance of a clear lab, cardiac, ophthalmologic, and dermatologic monitoring regimen as side effects can be rapid and severe. Prospective studies with encorafenib/binimetinib in BRAF-mutant primary CNS tumors are underway, and we eagerly anticipate their RESULTS:

Whole exome and transcriptome sequencing of colorectal and pancreatic cancer.

e15666 Background: Integration of Whole Exome Sequencing (WES) into clinical cancer therapeutics has revolutionized medicine in recent years. DNA sequencing alone may miss clinically actionable variants or identify aberrations that are not being transcribed. In this study we investigated the utility of integrating DNA and RNA sequencing in clinical samples. Methods: A cohort of 32 patient samples were analyzed by WES and RNA sequencing. Differential expression analysis was performed using a cohort of controls. Pathway analysis was performed using Ingenuity Pathway Analysis. WES and RNA analysis detected alterations including SNVs, indels, copy number events, fusions, alternate transcripts, TMB, MSI status and differential expression. Results: A total of 25 CRC (39-78yrs) and 7 pancreatic cancers (PCs) (51-91 yrs) were profiled by WES and RNA seq. A 66 yr old pt with neoplasm of rectosigmoid junction tumor was found to be KRAS wildtype and was treated with cetuximab plus FOLFIRI. Patient failed therapy after 2 yrs and sequencing revealed MET amplification which is a known mechanism of resistance to cetuximab treatment. Further, RNA expression analysis showed 44-fold increase in MET expression along with overexpression of AREG and EREG. Out of the 7 PCs, 3 cases that did not harbor KRAS mutation were found to harbor VTCN1/NRG1 fusion, FGFR1/G3BP2 fusion and BRAF V600E mutation respectively. A 50 year-old stage IV metastatic (met) PC pt was treated with standard of care regimens. Upon relapse the sample was found to harbor VTCN1/NRG1 fusion along with a TERT promoter mutation. RNA expression analysis revealed 54-fold increased expression of NRG1 which may lead to clinical trial enrollment. A 52 year-old male met Stage IV PC, was treated with rucaparib and irinotecan based on prior sequencing data. Upon relapse, the pt went on ATR inhibitor (BAY1895344) and progressed very quickly. Sequencing of the met lesion was found to harbor FGFR1/G3BP2 fusion which was also confirmed by RNA expression. A 55 year-old met pt with PC was treated with standard chemotherapy. Upon disease progression pt was sequenced and based on presence of BRAF V600E, pt was treated with trametinib and dabrafenib. Following disease progression on BRAF/MEKi, met sample was resequenced and RNA expression analysis found increased expression of MET, MACC1 and SMAD7 and 4-fold decrease in PTEN which could potentially cause resistance to BRAF/MEKi therapy. Conclusions: Our study highlights the utility of comprehensive testing integrating genomic and transcriptomic data, in identifying targeted therapy options for cancer patients.

Hairy cell leukemia with CCND1/IGH fusion gene and BRAF V600E mutation

A 75-year-old male evaluated for pancytopenia. Abnormal lymphocytes with hairy projections noted on peripheral blood. Bone marrow examination showed diffuse proliferation of CD20+ B-lymphocytes. Flowcytometry revealed monoclonal lambda-restricted B-cells expressing CD19, CD20, CD11c, CD103, CD25 and CD123, negative for CD5 and CD10. Additional staining showed positivity for cyclin-D1, Annexin-A1 and TRAP. FISH identified t(11;14). PCR was positive for BRAF V600E. Given the above findings, nonspecificity of t(11;14) and the presence of BRAF V600E; the diagnosis of HCL was favored. Patient achieved CR with infusional cladribine. Herein, we report the co-occurrence of CCND1/IGH and BRAF V600E in HCL, a rare scenario that could characterize a new subtype of HCL.

THE RELATIONSHIP OF THE BRAF-V600E MUTATION WITH THE EXPRESSION OF TRANSCRIPTIONAL, GROWTH FACTORS, COMPONENTS OF THE AKT / M-TOR SIGNALING PATHWAY IN THE TISSUE OF PAPILLARY THYROID CANCER

One of the initiating mutations in the development of thyroid cancer is BRAF-V600E, which leads to the various signaling cascades activation and changes in the production of transcription and growth factors. It is known that papillary thyroid cancer is characterized by activation of the expression of transcription factors NF-kB and HIF-2a and AKT/m-TOR signaling cascade. However, the relationship of the studied molecular markers in patients with the wild and mutant BRAF gene has not yet been studied. The aim of the work was to study the expression of transcription factors NF-65B p65 and NF-kB p50, HIF-1a, HIF-2a, growth factors VEGF, CAIX and VEGFR2, components of the AKT/m-TOR signaling pathway in patients with thyroid papillary cancer depending on the presence of mutations BRAF-V600E. Material and methods. It was included 40 patients with papillary thyroid cancer with the stage of the tumor process T1-4N0-2M0. The expression of the indicators was determined by real-time PCR. BRAF-V600E mutation was revealed by allele-specific PCR in real-time. Results and discussion. It was found that patients with the absence and presence of the BRAF-V600E mutation had similar clinical and morphological parameters of the disease, which was accompanied by a change in the molecular-biological characteristics of the tumor. The presence of the mutant form of the gene in the tumor led to a decrease in the AKT, cRAF, GSK-3P kinases mRNA levels and the overexpression of NF-kB p65, HIF-1 and VEGF. Conclusion. Patients with papillary thyroid cancer have no differences in the clinical and morphological characteristics of the disease, depending on the status of the BRAF-V600E gene, is characteristic. It was identified biological parameters associated with this somatic mutation. An increase in the mRNA level of growth and transcription factors was observed with a decrease in the activity of the AKT / m-TOR signaling pathway.

Molecular Profile and Clinicopathologic Features of Follicular Variant Papillary Thyroid Carcinoma.

The non-invasive encapsulated follicular variant of papillary thyroid carcinoma (FVPTC) has an indolent clinical behavior. Recently, it was proposed that this tumor type should be reclassified as non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). To characterize NIFTPs, we evaluated the molecular and clinicopathologic characteristics of each FVPTC subtype. This study enrolled 29 patients with FVPTC who underwent thyroidectomy between January 2007 and June 2017. They were classified as non-invasive encapsulated FVPTC (NIFTP, n = 10), invasive encapsulated FVPTC (n = 11), and infiltrative FVPTC (n = 8) by two independent pathologists. Genetic alterations were analyzed by targeted next-generation sequencing using formalin-fixed, paraffin-embedded tissue samples and the clinicopathologic characteristics were retrospectively reviewed. There was no difference in preoperative cytologic classification between NIFTPs and invasive encapsulated FVPTCs, whereas infiltrative FVPTC was more likely to be Bethesda class VI than the encapsulated type (50% versus 9.5%; P = 0.033). Lymph node metastasis was not found in NIFTPs. There was no BRAFV600E mutation in NIFTPs, whereas one of 11 invasive encapsulated FVPTCs and three of 8 infiltrative FVPTCs harbored BRAFV600E. RAS mutations were frequently detected in encapsulated FVPTCs (5 of 10 NIFTPs and 4 of 11 invasive encapsulated FVPTCs) but were only detected in one case of the infiltrative type. There were no differences in molecular or clinicopathologic profiles between non-invasive and invasive encapsulated FVPTCs, except for lymph node metastasis and the presence of BRAFV600E. NIFTP has favorable pathologic characteristics with a high frequency of RAS mutations.

Abstract 2762: Identification of novel immunotherapeutic targets in anaplastic thyroid cancer

Abstract Thyroid cancer is the most rapidly increasing cancer in the US with anaplastic thyroid cancer (ATC) being the rarest and most aggressive form. ATC patients display a higher mutational burden and are refractory to current mainstream treatments including second generation kinase inhibitors like vemurafenib (PLX4032). BRAFV600E is one of the most common mutations associated with this phenotype. Presence of BRAFV600E has been associated with an immunosuppressive microenvironment in thyroid cancer that presumably facilitates immune evasion. With recent advancements in immunotherapy, well defined targeted treatment plans can address the unmet medical need of ATC. In an effort to define potential immunotherapeutic targets that can be combined with small molecule inhibitors in this subtype, we evaluated the expression of immune checkpoint molecules in four thyroid cancer cell lines TPC-1 (papillary), BCPAP (BRAFV600E positive papillary), 8505C (BRAFV600E positive anaplastic) and CGTH-W-1 (follicular) by qRT PCR at the basal level and after treatment with vemurafenib and mTORC1 inhibitor rapamycin. Prominent co-stimulatory molecules like CD27, CD30, 4-1BB and DR3 were significantly downregulated at the transcript level in ATC (8505C) as compared to the other thyroid cancer subtypes. We observed a higher baseline expression of co-inhibitory molecules like full length CTLA4 (mCTLA4), soluble CTLA4 (sCTLA4), LAG3, 2B4, PD-1 and PD-L1 in ATC (8505C) compared to the other subtypes of thyroid cancer. Although vemurafenib treatment decreased the expression of PD-L1 as expected, interestingly, it enhanced the expression of co-inhibitory molecules LAIR1, 2B4, mCTLA4 and PD-1 by approximately 1.5 folds. These results suggest an inherent redundancy in inhibitory immune checkpoint molecules that presumably compensate for each other, functionally, making them novel targets in ATC. With our study we identified a distinct group of co-inhibitory molecules in ATC that can be targeted by antagonistic antibodies in a combinatorial treatment regimen with small molecule inhibitors like vemurafenib. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Ameet Kamat, Craig Berzofsky, Cameron Budenz, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Identification of novel immunotherapeutic targets in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2762.

CNS Langerhans cell histiocytosis: Common hematopoietic origin for LCH-associated neurodegeneration and mass lesions.

Central nervous system Langerhans cell histiocytosis (CNS-LCH) brain involvement may include mass lesions and/or a neurodegenerative disease (LCH-ND) of unknown etiology. The goal of this study was to define the mechanisms of pathogenesis that drive CNS-LCH. Cerebrospinal fluid (CSF) biomarkers including CSF proteins and extracellular BRAFV600E DNA were analyzed in CSF from patients with CNS-LCH lesions compared with patients with brain tumors and other neurodegenerative conditions. Additionally, the presence of BRAFV600E was tested in peripheral mononuclear blood cells (PBMCs) as well as brain biopsies from LCH-ND patients, and the response to BRAF-V600E inhibitor was evaluated in 4 patients with progressive disease. Osteopontin was the only consistently elevated CSF protein in patients with CNS-LCH compared with patients with other brain pathologies. BRAFV600E DNA was detected in CSF of only 2/20 (10%) cases, both with LCH-ND and active lesions outside the CNS. However, BRAFV600E+ PBMCs were detected with significantly higher frequency at all stages of therapy in LCH patients who developed LCH-ND. Brain biopsies of patients with LCH-ND demonstrated diffuse perivascular infiltration by BRAFV600E+ cells with monocyte phenotype (CD14+ CD33+ CD163+ P2RY12- ) and associated osteopontin expression. Three of 4 patients with LCH-ND treated with BRAF-V600E inhibitor experienced significant clinical and radiologic improvement. In LCH-ND patients, BRAFV600E+ cells in PBMCs and infiltrating myeloid/monocytic cells in the brain is consistent with LCH-ND as an active demyelinating process arising from a mutated hematopoietic precursor from which LCH lesion CD207+ cells are also derived. Therapy directed against myeloid precursors with activated MAPK signaling may be effective for LCH-ND. Cancer 2018;124:2607-20. © 2018 American Cancer Society.