BRAF Point Mutations Research Articles

Emerging RAS, BRAF, and EGFR mutations in cell-free DNA of metastatic colorectal patients are associated with both primary and secondary resistance to first-line anti-EGFR therapy.

Oncogenic RAS mutations are negative biomarkers of response to epidermal growth factor receptor (EGFR) blockade. RAS mutations are usually detected in biopsies of primary colorectal tumors. However, the genomic profiles of primary tumors and metastases are not always concordant, and chemotherapeutic agents can alter the tumor molecular landscape. Cell-free DNA (cfDNA) is a novel tool to detect molecular heterogeneity. This study evaluated the clinical utility of cfDNA to predict primary or secondary resistance to EGFR blockade in patients with metastatic colorectal cancer. Thirty metastatic colorectal cancer patients without RAS and BRAF mutations were prospectively enrolled and treated with cytotoxic agents and EGFR blockade as first-line therapy. cfDNA was analyzed for the presence of RAS, BRAF, and EGFR (S492R) point mutations before initiating chemotherapy and every 2months during chemotherapy. The analysis was performed in 223 plasma samples from all 30 patients. Of the 30 patients, five had RAS mutations in their cfDNA before starting chemotherapy and did not respond. Twenty-four of the remaining 25 patients without cfDNA RAS mutations had a response. Twenty of the 24 responders developed secondary resistance and cfDNA RAS mutations were found in 17 of the 20. cfDNA BRAF mutations were found in seven, and EGFR mutations were found in eight of the 20 patients. Emerging RAS, BRAF, and EGFR mutations occurred in patients with primary and secondary resistance to EGFR blockade. The detection of these mutations in cfDNA is a promising approach to predict treatment response and secondary resistance.

MiRNA expression can classify pediatric thyroid lesions and increases the diagnostic yield of mutation testing.

Genetic alterations in multiple cell signaling pathways are involved in the molecular pathogenesis of thyroid cancer. Oncogene mutation testing and gene-expression profiling are routinely used for the preoperative risk management of adult thyroid nodules. In this study, we evaluated the potential value of miRNA biomarkers for the classification of pediatric thyroid lesions. Double-blind case-control study with 113 resected pediatric lesions: 66 malignant and 47 benign. Quantitative and qualitative molecular data generated with a 10-miRNA expression panel (ThyraMIR) and a next-generation sequencing oncogene panel (ThyGeNEXT) were compared with clinicopathological parameters. miRNAs were differentially expressed in benign versus malignant tumors with distinct expression patterns in different histopathology categories. The 10-miRNA classifier identified 39 (59%) malignant lesions with 100% specificity. A positive classifier score was associated with lymph node metastasis, extrathyroidal extension and intrathyroidal spread. Genetic alterations associated with increased risk for malignancy were detected in 35 (53%) malignant cases, 20 positive for point mutations in BRAF, HRAS, KRAS, NRAS, PIK3CA, or TERT and 15 positive for gene rearrangements involving ALK, NTRK3, PPARG, or RET. The 10-miRNA classifier correctly identified 11 mutation-negative malignant cases. The performance of the combined molecular test was 70% sensitivity and 96% specificity with an area under the curve of 0.924. These data suggest that the regulatory miRNA pathways underlying thyroid tumorigenesis are similar in adults and children. miRNA expression can identify malignant lesions with high specificity, augment the diagnostic yield of mutation testing, and improve the molecular classification of pediatric thyroid nodules.

Presence and prevalence of UV related genetic mutations in uveal melanoma: similarities with cutaneous melanoma.

Ultraviolet (UV) radiation is an accepted etiological factor in cutaneous melanoma (CM), however its role in uveal melanoma (UM) is controversial. Partly as a consequence, CM and UM are often considered to be separate conditions, and advances in the treatment of CM have not led to joint clinical trials or parallel improvements in survival of UM. This study hypothesized that a subset of UM tumors displays evidence of genetic changes consistent with UV-related damage similar to that shown in CM. Analysis of the Broad Institute's Firebrowse depository of 80 UM samples and 343 CM samples, together with the Sanger Institute's Catalogue of Somatic Mutations in Cancer depository of 995 UM and 12,447 CM samples was undertaken to identify the most frequently mutated genes, mutation types, and specific nucleotide variants (SNVs) in each condition. Somatic mutation data were cross-correlated and shared mutations assessed against known effects of UV radiation. The proportion of samples with C>T substitutions (a classic genetic marker of UV-related damage) was higher in UM than CM on both DNA strands (17.0% vs 13.1%, p=0.038). The most frequently encountered cross-correlated mutated genes between UM and CM were, in order, BRAF, NRAS, TP53, CDKN2A, TERT, PTEN, ARID2, and KMT2C, with multiple common BRAF point mutations. Each cross-correlated mutation, and each common point mutation in BRAF, was associated with UV-related mechanistic changes. These findings support the hypothesis that the etiology of a substantial minority of UMs may be more UV dependent than previously recognized.

BRAF molecular testing in cytopathology: Implications for diagnosis, prognosis, and targeted therapeutics.

BRAF molecular testing in cytopathology: Implications for diagnosis, prognosis, and targeted therapeutics.

CIC Mutation as a Molecular Mechanism of Acquired Resistance to Combined BRAF-MEK Inhibition in Extramedullary Multiple Myeloma with Central Nervous System Involvement.

Combined MEK-BRAF inhibition is a well-established treatment strategy in BRAF-mutated cancer, most prominently in malignant melanoma with durable responses being achieved through this targeted therapy. However, a subset of patients face primary unresponsiveness despite presence of the activating mutation at position V600E, and others acquire resistance under treatment. Underlying resistance mechanisms are largely unknown, and diagnostic tests to predict tumor response to BRAF-MEK inhibitor treatment are unavailable. Multiple myeloma represents the second most common hematologic malignancy, and point mutations in BRAF are detectable in about 10% of patients. Targeted inhibition has been successfully applied, with mixed responses observed in a substantial subset of patients mirroring the widespread spatial heterogeneity in this genomically complex disease. Central nervous system (CNS) involvement is an extremely rare, extramedullary form of multiple myeloma that can be diagnosed in less than 1% of patients. It is considered an ultimate high-risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. Here we not only describe the first patient with an extramedullary CNS relapse responding to targeted dabrafenib and trametinib treatment, we furthermore provide evidence that a point mutation within the capicua transcriptional repressor (CIC) gene mediated the acquired resistance in this patient. KEY POINTS: BRAF mutations constitute an attractive druggable target in multiple myeloma. This is the first genomic dissection of the central nervous system involvement in a multiple myeloma patient harboring a druggable BRAFV600E mutation. Deep genomic characterization of the extramedullary lesion prompted a personalized therapeutic approach. Acquisition of CIC mutation confers a mechanism of BRAF-MEK inhibitor drug resistance in multiple myeloma. The in silico interrogation of the CoMMpass clinical study revealed 10 patients with somatic mutations of CIC and its downregulation at gene expression level in multiple myeloma. CIC gene silencing decreases the sensitivity of multiple myeloma cells to BRAF-MEK inhibition in vitro. The correlation between CIC downregulation and ETV4/5 nuclear factor expression in multiple myeloma BRAF-mutant cells is shown for the first time. CIC mutation, its downregulation, and the related downstream effect on MMP24 support disseminative potential providing new clues in the extramedullary biology definition.

Deep Small RNA Sequencing of BRAF V600E Mutated Papillary Thyroid Carcinoma With Lymph Node Metastasis.

Papillary thyroid carcinoma (PTC) is the most predominant subtype of thyroid cancer, contributing to more than 80% of all thyroid or endocrine malignancies. Its prognosis is relatively good compared to other cancers, with more than 90% overall 10-year survival rate (Haugen et al., 2016). However, despite the favorable prognosis, some cases exhibit aggressive phenotype. On average, 50% of the patients are presented with lymph node metastases (LNM) at diagnosis (Saiselet et al., 2015). LNM in PTC confers various poor prognostic indicators; it increases recurrence risk and decreases long term survival predominantly in patients older than 45 years old (Lundgren et al., 2006; Xing, 2013). Treating recurrent PTCs is still a challenge (Pasternak and Shen, 2017) and one of the important issues yet to be solved in PTC patient management is the mortality and morbidity associated with the recurrent disease (Lee et al., 2013). MicroRNAs (miRNAs) are short, endogenous noncoding RNAs first identified in Caenorhabditis elegans which regulate gene expression by binding to the 3′-UTR of target messenger RNA (mRNAs). Their post-transcriptional regulatory functions are involved in controlling the level of proteins involved in numerous biological processes, including embryogenesis, organogenesis, tissue homeostasis, immune system function and cell cycle control (Tafrihi and Hasheminasab, 2019). Relationship between miRNAs and tumor growth, tumor progression and metastasis has been demonstrated by many studies, indicating the ability of these molecules to be used as biomarkers for diagnosis and prognosis (Iorio and Croce, 2012). MiRNA also plays a role as biomarker in predicting lymph nodes metastasis (LNM) in thyroid cancer (Ab Mutalib et al., 2016; Mutalib et al., 2016; Mohamad Yusof et al., 2018). P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a quite recently discovered class of RNAs and more than 30,000 piRNA genes been identified within the human genome (Zhang et al., 2014; Wang et al., 2019). Originally described as key regulators for the germline maintenance and transposon silencing, these short RNAs were disregarded for a long time due to our limited knowledge regarding their function (Weng et al., 2019). However, emergent of new data reveals that unusual expression of piRNAs is a distinct feature Fin many diseases, including cancers (Weng et al., 2019). This has changed our perspectives on their significance in various diseases. Epigenetically, piRNAs are also the post-transcriptional regulators of the expression of specific downstream target genes (Watanabe and Lin, 2014), and accumulating evidence demonstrates that similarly to miRNAs, piRNAs also possess both oncogenic and tumor-suppressive roles (Weng et al., 2019). BRAF is a serine-threonine kinase that is activated by RAS binding and protein recruitment to the cell membrane. Activation of MEK along with the MAPK signaling pathway is activated by BRAF phosphorylation (Nikiforov and Nikiforova, 2011). The most frequent genetic changes in PTC are point mutations of BRAF which are observed in 35 to 70% of PTC cases (Li et al., 2012; Xing, 2013). More than 95% of BRAF mutations in thyroid cancers are thymine to adenine transversion at position 1799 (T1799A) which result in the substitution of glutamate from valine at residue 600 (V600E) (Xing et al., 2015). Various studies have also shown that the BRAF V600E mutation is related to LNM, as further presented by a meta-analysis by Song et al. (2018). In the past years, the evolution of next-generation sequencing technologies has enabled global transcriptome expression profiling and the discovery of novel human miRNAs (Kozomara and Griffiths-Jones, 2011; Kozomara and Griffiths-Jones, 2014; Kozomara et al., 2019) and piRNAs (Weng et al., 2019). Coincidentally, PTC has a relatively lower overall mutation burden hence termed as quiet genome (Cancer Genome Atlas Research Network, 2014; Siraj et al., 2016), therefore investigating the regulatory molecules such as the miRNAs and piRNAs has the potential to deepen our understanding of this cancer. This Data Report aims to provide the readers a comprehensive dataset of small RNAs expression derived from next-generation sequencing in an unbiased manner. For detailed analysis of this dataset focusing on miRNAs with biological insights in PTC, please refer to our original article published in Frontiers of Endocrinology (Mohamad Yusof et al., 2018).

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification.

We have developed a method for spatially resolved genetic analysis of formalin-fixed paraffin-embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 μm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF-7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification

AbstractWe have developed a method for spatially resolved genetic analysis of formalin‐fixed paraffin‐embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 μm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF‐7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Molecular evaluation of BRAF gene mutation in thyroid tumors: Significant association with papillary tumors and extra thyroidal extension indicating its role as a biomarker of aggressive disease

BackgroundSomatic mutation of the BRAF gene is one to be the most commonly known genetic change in thyroid tumors especially papillary thyroid cancers. The T1799A activating point mutation is detected in >98% of the thyroid tumors, and result in substitution of amino acid valine at position 600 to glutamic acid. MethodIn this study, we evaluated BRAF mutation in 95 Indian thyroid tumors by pyrosequencing assay. ResultsOverall, 36 cases (38%) showed presence of BRAF V600E mutation, while none of the cases showed V600 K mutation. BRAF mutation was found predominantly in female patients in comparison to males (38.4% vs. 36.4%, p = .86). Likewise, smaller sized tumors (≤2.0 cm) showed increased frequency of BRAF mutation as compared to larger sized tumors which were greater than equal to 2 cm (46% vs. 34.4%, p = .64). Furthermore, the frequency of BRAF mutations was significantly higher in conventional PTC tumor type in comparison to non-conventional and other than PTC tumor type (56% vs. 35% vs. 4%, p = .0007). Notably, a significant correlation between presence of BRAF mutation and extra-thyroidal extension was noted. Nevertheless, presence of BRAF mutation was neither associated with capsular/vascular invasion, nor with tumor necrosis. ConclusionsPyrosequencing assay was found to be highly sensitive and accurate method for detecting BRAF point mutations. The frequency and distribution pattern of BRAF mutations is similar to global reports. Furthermore, association of BRAF mutation with extra thyroidal extension indicates its aggressive nature and thus can provide insights into the progression of thyroid tumors from less aggressive to poorly differentiated subtype.

LGG-26. TYPE II BRAF INHIBITOR TAK-580 SHOWS PROMISE FOR UPCOMING CLINAL TRIAL AS EVIDENCED BY SINGLE PATIENT IND STUDY

Abnormal signaling of the RAS/RAF pathway predominates in pediatric low-grade gliomas (PLGGs). Most (85%) non-NF1 low-grade gliomas have RAS-independent BRAF activation, either due to KIAA1549:BRAF truncated fusion duplication or V600E point mutation. We previously identified two different type II BRAF inhibitors capable of inhibiting downstream activation of pERK and tumor progression in vivo for multiple point mutations and BRAF truncated fusion forms. One compound, in particular, known as TAK-580 was active on authentic human PLGG cells ex vivo and showed excellent CNS penetration. We conducted a single patient IND study for a patient with refractory, progressive LGG. Patient was initially diagnosed at age 2 years and treated with 9 different chemotherapy regimens up until age 15 years prior to starting TAK-580. Study drug was begun orally on a QOD schedule; one cycle was comprised of 28 days. Based on both tolerance and pharmacokinetic analyses, the treatment schedule and dosing were adjusted to weekly, comparable to the adult dosing schedule. MR imaging of her tumor burden has remained stable, and patient has recently started her 33rd cycle of once weekly TAK-580. TAK-580 was well tolerated. Grade 3 and 4 likely or possibly related toxicities included acneiform rash, hyperglycemia, elevated creatinine kinase, elevated ALT and decreased phosphorus. TAK-580 posess a unique mechanism of action targeting the BRAF/MEK complex and inhibiting both BRAF point mutations and common structural abnormalities in PLGGs. These initial results support the upcoming phase I/0/II trial of TAK-580 for children with LGG and other RAS/RAF/MEK/ERK pathway activated tumors.

Anaplastic lymphoma kinase (ALK) gene rearrangements in radiation‐related human papillary thyroid carcinoma after the Chernobyl accident

Childhood radiation exposure has been associated with increased papillary thyroid carcinoma (PTC) risk. The role of anaplastic lymphoma kinase (ALK) gene rearrangements in radiation‐related PTC remains unclear, but STRN‐ALK fusions have recently been detected in PTCs from radiation exposed persons after Chernobyl using targeted next‐generation sequencing and RNA‐seq. We investigated ALK and RET gene rearrangements as well as known driver point mutations in PTC tumours from 77 radiation‐exposed patients (mean age at surgery 22.4 years) and PTC tumours from 19 non‐exposed individuals after the Chernobyl accident. ALK rearrangements were detected by fluorescence in situ hybridisation (FISH) and confirmed with immunohistochemistry (IHC); point mutations in the BRAF and RAS genes were detected by DNA pyrosequencing. Among the 77 tumours from exposed persons, we identified 7 ALK rearrangements and none in the unexposed group. When combining ALK and RET rearrangements, we found 24 in the exposed (31.2%) compared to two (10.5%) in the unexposed group. Odds ratios increased significantly in a dose‐dependent manner up to 6.2 (95%CI: 1.1, 34.7; p = 0.039) at Iodine‐131 thyroid doses >500 mGy. In total, 27 cases carried point mutations of BRAF or RAS genes, yet logistic regression analysis failed to identify significant dose association. To our knowledge we are the first to describe ALK rearrangements in post‐Chernobyl PTC samples using routine methods such as FISH and IHC. Our findings further support the hypothesis that gene rearrangements, but not oncogenic driver mutations, are associated with ionising radiation‐related tumour risk. IHC may represent an effective method for ALK‐screening in PTCs with known radiation aetiology, which is of clinical value since oncogenic ALK activation might represent a valuable target for small molecule inhibitors.

Abstract A129: A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor

Abstract BRAF V600E drives tumors by hyperactivating ERK signaling, is activated in a RAS-independent manner, and signals as a monomer. Selective inhibitors of RAF potently inhibit BRAF V600E and downstream ERK signaling, and have been associated with impressive clinical responses in patients whose tumors harbor V600E mutations in BRAF. Almost all patients, however, ultimately develop resistance. To date, acquired resistance mechanisms validated in patient samples have included splice forms of BRAF, mutations in NRAS, and BRAF amplification, all of which promote formation of RAF dimers, and mutations in MEK. Second-site point mutations in BRAF that promote dimer formation and are associated with acquired resistance have not previously been identified in RAF inhibitor-resistant tumors. Here, we report a complete response followed by clinical progression of a BRAF V600E-mutant pediatric brain tumor treated with the RAF inhibitor dabrafenib. We identified a second-site mutation in BRAF at the time of progression that was not present in the pretreatment tumor. Further study indicated the acquired mutation is in cis with V600E. We demonstrate that the acquired resistance mutation induces RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Moreover, we showed that ERK signaling activated by the double mutation is sensitive to MEK inhibition or to the RAF plus and MEK inhibitor combination. We also find that two novel RAF dimer inhibitors may overcome resistance mediated by the novel mutation in BRAF. This is the first study, to our knowledge, that demonstrates the emergence of a confirmed second-site mutation in BRAF V600E and validates that this mutation is responsible for acquired resistance in an initially responding patient treated with dabrafenib. The mechanism of acquired resistance can be abrogated by the novel RAF dimer inhibitors. Our data confirm that a novel class of RAF dimer inhibitors are active against an acquired resistance mutation, suggesting an improved personalized treatment option for patients who harbor similar second-site mutations in BRAF. Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, David Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A129.

Rapid Clinical and Radiographic Response With Combined Dabrafenib and Trametinib in Adults With BRAF-Mutated High-Grade Glioma.

BRAF V600E mutations have been successfully treated with targeted therapy in melanoma, non-small cell lung cancer, and thyroid cancer. Interestingly, these mutations have also been identified in a subset of pediatric and adult brain tumors, with several cases reportedly responding to targeted therapy. However, these reports have been limited to single-agent BRAF inhibitor therapy and recurrent disease. Herein, we report dramatic clinical and radiographic responses to combination dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) in 2 adults with high-grade gliomas (HGGs), with 1 patient treated in the first-line setting. These observations, together with prior case reports, advocate for routine screening of BRAF point mutations in adult HGGs, and suggest that treatment with dual-targeted therapy, even in newly diagnosed cases, is safe and effective.

Genetic evidence for the role of ultraviolet radiation in the pathogenesis of uveal melanoma

PurposeRecent advances in the understanding and treatments of metastatic cutaneous melanoma (CM) have not led to parallel improvements in the care of metastatic uveal melanoma (UM), and consequently, the two conditions are now often viewed as separate entities. One possible difference is the aetiological role of ultraviolet (UV) radiation, which although well‐established in CM, remains uncertain in UM. This study hypothesised that UV radiation is a pathogenic factor in UM development, evidenced by genetic changes consistent with UV‐related damage in UM.MethodsWe analysed data from 993 UM patient samples and 11803 CM patient samples available from the Catalogue of Somatic Mutations in Cancer (COSMIC) as well as 80 UM patient samples and 343 CM patient samples from the Broad Institute GDAC FireBrowse. UM samples were probed to identify the most frequently mutated genes, mutation types and specific nucleotide substitutions. Somatic mutation data was then cross‐correlated with CM samples from COSMIC and Broad Institute GDAC FireBrowse.ResultsThe most common overlapping mutated genes were BRAF, PTEN, CDKN2A, TERT, NRAS, TP53 and ARID2, with four shared point mutations in BRAF (V600E (1799T>A)), NRAS (Q61R (182A>G)) and TP53 (R273C (817C>T)), R248Q (743G>A)). These gene mutations were found to be strongly associated with UV‐related damage in previous scientific reports. The proportion of samples with C>T substitutions (a marker of UV‐related damage) were similar between UM and CM on both DNA strands (20.35% vs 20.05%) and coding strand (11.3% vs 15.7%).ConclusionsThese findings support the hypothesis that the pathogenesis of UM is more dependent on UV radiation than previously thought.

Mutational Status of NRAS and BRAF Genes and Protein Expression Analysis in a Series of Primary Oral Mucosal Melanoma.

Primary oral mucosal melanoma is an extremely rare and aggressive tumor arising from melanocytes located in the mucosal epithelium of the oral cavity. Although malignant melanoma of oral mucosa shares some clinical features with its cutaneous counterpart, it has been associated with a worst prognosis; its etiopathogenesis are still only partially unraveled as there is no influence of UV radiation. It is known that the mitogen-activated protein kinase pathway mediates cellular responses to growth signals and its activation is an important phenomenon in melanoma. The aim of this study was to evaluate NRAS and BRAF genes, both components of mitogen-activated protein kinase molecular pathway, and compare with their protein expression. Point mutations of NRAS (codons 12, 13, and 61) and BRAF (codon 600) were screened by pyrosequencing method, and its results were associated to the protein expression of RAS and BRAF performed by immunohistochemistry. The authors observed mutation in BRAF 600 (3/14), NRAS codons 12 and 13 (2/14), and NRAS codon 61 (2/8). One case showed positive RAS protein expression, but no mutation was observed. Twelve in 14 cases showed positive BRAF protein expression: 3 cases showed BRAF mutation; 2 cases showed NRAS codon 61 mutation; 2 cases showed NRAS codons 12 and 13 mutation but not simultaneously. Although NRAS and BRAF mutation frequency and RAS protein expression are low, BRAF protein expression was intense; probably, NRAS and BRAF mutations are independent events and alternative molecular mechanisms in the primary oral mucosal melanoma tumorigenesis.

Single Point Mutations in Pediatric Differentiated Thyroid Cancer.

Differentiated thyroid cancer (DTC) is rare in children. Previous studies have suggested that it has different clinicopathologic features and mutation profiles compared with adult DTC. However, those studies focused on a single or limited number of gene mutations. This study comprehensively investigated a large series of pediatric DTC for single point mutations in BRAF, HRAS, KRAS, NRAS, PIK3CA, PTEN, and TERT. It also analyzed associations between clinicopathologic features and the BRAFV600E mutation. Eighty-nine consecutive cases seen in children and adolescents (≤18 years) during 1998-2015 were identified. Rare variants of DTC were excluded, and the study focused on 72 (91.1%) classical papillary thyroid carcinoma (PTC) and seven (8.9%) follicular variant PTC. These included 68 (86.1%) females and 11 (13.9%) males, with a median age of 15.5 years (range 8-18 years). The clinical and histopathological data were obtained from medical records. DNA was extracted from paraffin-embedded tumor tissue, and was PCR-amplified and directly sequenced. Mutations detected included BRAFV600E in 19/72 (26.4%) classical PTC samples, and in none of seven follicular variant PTC. Other mutations included: 1/78 (1.3%) successfully amplified tumor samples with TERT C228T; 2/79 (2.5%) NRAS 61 (c.181C>A and c.182A>G); 1/73 (1.4%) PIK3CA exon 9 (c.1589A>G and c.1598C>T in one tumor); 1/79 (1.3%) PIK3CA exon 20 (c.2951G>A); and 1/74 (1.4%) PTEN exon 5 (c.295G>A). No mutation was found in HRAS, KRAS, NRAS12, PTEN exons 6, 7, and 8, and TERT C250T. No significant association was found between BRAFV600E mutation and sex, extrathyroidal invasion, tumor multifocality, vascular invasion, lymph node or distant metastases, and persistent/recurrent disease. In pediatric DTC, the prevalence of the BRAFV600E mutation is significantly less common compared with adult DTC, and there is no association between this mutation and the histopathological features and outcome of PTC. PIK3CA, PTEN, NRAS 61, and TERT C228T mutations are rare.

Nuclear morphometric findings in undetermined cytology: A possible clue for prediction of BRAF mutation in papillary thyroid carcinomas

ABSTRACTPurpose: To investigate the possible relationship between the nuclear morphometric characteristics (nuclear perimeter, roundness, nuclear area, and nuclear shape) and BRAF mutation status in papillary thyroid carcinoma cases with a prior diagnosis of undetermined cytology. Materials and methods: Total thyroidectomy specimens obtained from 48 patients with papillary thyroid carcinoma with a prior diagnosis of undetermined cytology were included. Morphometric analysis under light microscopy included measurements of the perimeter, shape factor, nuclear area, and roundness of thyrocyte nuclei from the cytological smear preparations. Mutational analysis, including immunohistochemistry and polymerase chain reaction, was performed in formalin fixed paraffin embedded tissue blocks. Results: BRAF V600E mutation was detected in 7 of 48 cases (14.5%). The nuclear perimeter and nuclear area in mutated cases were significantly higher than the wild type (p = 0.005). Shape factor (p = 0.681) and roundness (p = 0.752) values did not significantly differ between the wild-type and mutant groups. No significant relationship was evident between BRAF expression and BRAF point mutation. Conclusion: In cases with positivity for BRAF mutation, the nuclear perimeter and nuclear area were significantly increased. These findings suggest that morphometric variables are predictive markers for papillary thyroid carcinoma cases with positivity for BRAF mutation. However, further trials on larger series are warranted to understand the significance and predictive value of nuclear morphometric analysis in these circumstances.

A Brief Review on The Molecular Basis of Medullary Thyroid Carcinoma.

Approximately 5-10% of all thyroid cancers are medullary thyroid carcinomas (MTC). MTC is mainly sporadic in nature, but 20-30% of cases are hereditary. Genetic testing for hereditary MTC is very important for the patient and his family, but the patients must be receiving appropriate genetic counseling. About 98% of patients with hereditary MTC have germline mutations in exons 10, 11, 13, 14, 15, 16 and intron 16 of the REarrangement during transfection (RET) proto-oncogene, but the etiology of the more frequent sporadic form of MTC (sMTC) is not well understood. Recently, it has been reported that apparently sporadic MTC may involve point mutations in BRAF and RAS genes, with an overall prevalence of almost 10%. Also alteration and abnormal expression of miRNA has been described in MTC. In this review, we attempted to mention some mutations and molecular changes in sporadic and hereditary MTC pathogenesis.

Classical point mutations of RET, BRAF and RAS oncogenes are not shared in papillary and medullary thyroid cancer occurring simultaneously in the same gland.

Papillary (PTC) and medullary (MTC) thyroid carcinomas represent two distinct entities, but quite frequently, they may occur simultaneously. To provide genetic analysis of PTC and MTC occurring in the same patient (PTC/MTC) to elucidate their origin. Sequencing analysis of RAS, BRAF and RET oncogenes hot spots mutations in tumoral and normal tissues of 24 PTC/MTC patients. Two of 24 patients (8.3%) were affected by familial MTC (FMTC) harboring RET germline mutations in all tissues. Eight of 22 (36.4%) sporadic cases did not show any somatic mutation in the three tissue components. Considering the MTC component, 10/22 (45.4%) patients did not show any somatic mutation, 7 of 22 (31.8%) harbored the M918T RET somatic mutation and 4/22 (18.2%) presented mutations in the H-RAS gene. In an additional case (1/22, 4.6%), H-RAS and RET mutations were simultaneously present. Considering the PTC component, 1 of 24 (4.2%) patients harbored the V600E BRAF mutation, 1 of 24 (4.2%) the T58A H-RAS mutation and 1 of 24 (4.2%) the M1T K-RAS mutation, while the remaining PTC cases did not show any somatic mutation. In one case, the MTC harbored a RET mutation and the PTC a BRAF mutation. None of the mutations found were present in both tumors. To our knowledge, this is the first study analyzing a possible involvement of RET, BRAF and RAS oncogene mutations in PTC/MTC. These data clearly suggest that the classical activating mutations of the oncogenes commonly involved in the pathogenesis of PTC and MTC may not be responsible for their simultaneous occurrence.

Abstract 485: Blood-based profiling of patients with NSCLC using CLIA certified cell-free DNA tests for EGFR, KRAS and BRAF point mutations and test use in clinical practice

Abstract Nearly 80% of patients will not have mutation results available at their initial oncology consult and as many as 1 in 4 patients will begin treatment in advance of receiving their tumor mutation results. These factors hinder diagnosis at the molecular level, which is an important component in the decision to commence treatment with specific targeted therapies. The goal of this study was to measure the utility of a CLIA-certified Laboratory Developed Test (LDT) in the absence of a tissue diagnosis. EGFR, KRAS and BRAF point mutations within the cell-free DNA (cfDNA) isolated from the plasma of patients with advanced stages of cancer were analyzed using Droplet Digital™ PCR (ddPCR) technology. The specific tests detected the EGFR sensitizing mutations L858R and exon 19 deletion (E746 - A750) and EGFR resistance mutation, T790M, the KRAS mutations G12C, G12V and G12D, as well as the BRAF V600E mutation. The tests were comprised of three components: (i) a whole-blood collection kit that ships at ambient temperature, (ii) cfDNA isolated from plasma for analysis using ddPCR, and (iii) a secure laboratory information management system (LIMS) for sample accessioning and report generation. Test mutation status results were reported within 72 hours of blood shipment from the physicians’ office. To date we have processed greater than 2000 individual variant tests for patients with NSCLC. The predominant tumor type for EGFR sensitizing mutations requested was NSCLC (95%). Interestingly, even BRAF test orders were > 90% from NSCLC patients. Additionally, the percentage of tests requested that were positive for each mutation category were 12% for EGFR sensitizing, 20% for EGFR resistance, 14% for KRAS and 1.8% for BRAF. Significantly, T790M test orders were observed to have increased five-fold over the last three months of testing. We have developed highly sensitive, 72 hour test to results blood-based assays as a part of the GeneStratTM panel, that expand the utility of laboratory testing for patients previously diagnosed with cancer. Citation Format: Hestia Mellert, Scott Thurston, Trudi Foreman, Westen Hahn, Nicholas Dupuis, Gary Pestano. Blood-based profiling of patients with NSCLC using CLIA certified cell-free DNA tests for EGFR, KRAS and BRAF point mutations and test use in clinical practice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 485.