BRAF Exon Research Articles

Whole Exome Sequencing of SMO, BRAF, PTCH1 and GNAS in Odontogenic Diseases.

Odontogenic diseases are diagnosed based on clinical course, imaging, and histopathology. However, a definitive diagnosis is not always possible. We analyzed whole exons of SMO, BRAF, PTCH1 and GNAS using next-generation sequencing (NGS) in 18 patients. Of the 6 patients with ameloblastoma, 2 patients had the same missense mutation in BRAF, and 1 patient with peripheral ameloblastoma had a missense mutation in PTCH1. Of the 7 patients with odontogenic keratocyst, 4 patients had a missense mutation in PTCH1, 2 patients had missense mutations in BRAF, and 1 patient had a missense mutation in SMO. The patient with odontoma had missense mutations in SMO, BRAF and PTCH1. One patient with cement-osseous dysplasia had missense mutations in SMO and PTCH1. The patient with adenomatoid odontogenic tumor had missense mutations in SMO. Whole exome sequencing of the above genes by NGS would be useful for the differential diagnosis of odontogenic diseases.

Mutational Profile of Driver Genes in Brazilian Melanomas.

PURPOSEMutation testing of the key genes involved in melanoma oncogenesis is now mandatory for the application of targeted therapeutics. However, knowledge of the mutational profile of melanoma remains largely unknown in Brazil.PATIENTS AND METHODSIn this study, we assessed the mutation status of melanoma driver genes BRAF, NRAS, TERT, KIT, and PDGFRA in a cohort of 459 patients attended at Barretos Cancer Hospital between 2001 and 2012. We used polymerase chain reaction followed by Sanger sequencing to analyze the hot spot mutations of BRAF exon 15 (V600E), NRAS (codons 12/13 and 61), TERT (promoter region), KIT (exons 9, 11, 13, and 17), and PDGFRA (exons 12, 14, and 18) in tumors. The mutational profile was investigated for associations with demographic, histopathologic, and clinical features of the disease.RESULTSThe nodular subtype was most frequent (38.9%) followed by the superficial spreading subtype (34.4%). The most frequent tumor location was in the limbs (50.0%). The mutation rates were 34.3% for TERT and 34.1% for BRAF followed by NRAS (7.9%), KIT (6.2%), and PDGFRA (2.9%). The BRAF (P = .014) and TERT (P = .006) mutations were associated with younger patients and with different anatomic locations, particularly in the trunk, for the superficial spreading and nodular subtypes, respectively (P = .0001 for both). PDGFRA mutations were associated with black skin color (P = .023) and TERT promoter mutations with an absence of ulceration (P = .037) and lower levels of lactate dehydrogenase. There was no association between patient survival rates and mutational status.CONCLUSIONThe similar mutational profile we observe in melanomas in Brazil compared with other populations will help to guide precision medicine in this country.

BRAFV600E-Positive Precursors As Molecular Markers of Bone Marrow Involvement in Pediatric Langerhans Cell Histiocytosis

Introduction: The BRAF mutation V600E, the most common somatic mutation in Langerhans cell histiocytosis (LCH), has been reported in approximately 50% of LCH patients and is associated with certain high-risk clinical features. Precursors harboring this mutation can differentiate into Langerhans cells resulting in infiltrates in multiple organs under inflammatory conditions. However, BRAF status in the bone marrow of pediatric LCH patients is unclear. The present study examined somatic mutations in paired tumor and bone marrow samples, using a highly sensitive assay involving next-generation targeted sequencing and droplet digital polymerase chain reaction (PCR) for pediatric LCH patients. Methods: Between 1996 and 2019, in total of 17 Japanese pediatric patients with LCH were enrolled. The male/female ratio was 7/11. Ages of onset of LCH were median 13 months (range 5-193 months). At diagnosis of LCH, 2 patients were positive for risk organ involvement, 15 were negative. We retrospectively performed mutational analyses of 17 LCH cases using formalin-fixed paraffin-embedded LCH tumor specimens to provide templates for PCR-based targeted amplicon sequencing with customized primers to detect mutations in exons 12 and 15 in BRAF, and exons 2 and 3 in MAP2K1. Thereafter, we identified somatic mutations in the 17 paired bone marrow samples via droplet digital allele-specific PCR, targeting BRAF V600E and BRAF exon 12 in-frame deletion 496-500 (Ex12 in-del). Results: We detected BRAF V600E in 11 of 17 tumor samples (65%) and the BRAF Ex 12 in-del in 3 of 17 tumors (18%). We identified BRAF V600E in bone marrow samples in 10 of the 11 cases (90%) with the mutation in the tumor at low variant allele frequency (median 0.25%, range 0.14-7.0%). BRAF Ex 12 in-del was not detected in the bone marrow. Cases with detectable bone marrow involvement included eight patients with multi-system disease affecting multiple organs, one patient with multi-focal bone disease, and one patient with single-system disease. Clinical phenotypes including relapse did not correlate with BRAF V600E upon detection in the bone marrow. Conclusion: We established the sensitive assay based on PCR-based targeted NGS for detecting somatic mutations in LCH even accessible for formalin-fixed, paraffin-embedded clinical specimens. Bone marrow involvement is frequently detectable at the molecular level in pediatric LCH with the BRAF V600E mutation. A prospective study is warranted to evaluate the clinical impact of mutational burden in bone marrow. Disclosures Kudo: Unum Therapeutics: Patents & Royalties. Imai:Juno Therapeutics: Patents & Royalties.

ANGI-05. ORIGIN OF MICROVASCULAR PROLIFERATION IN PILOCYTIC ASTROCYTOMA - MVP IN PILOCYTIC ASTROCYTOMA MIGHT PARTIALLY COMPRISE TUMOR-DERIVED CELLS

Abstract Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVP in PA contained tumor-derived cells. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was characterized by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP may be tumor-derived.

Impact of ultrasonographic features, cytomorphology and mutational testing on malignant and indeterminate thyroid nodules on diagnostic accuracy of fine needle cytology samples: A prospective analysis of 141 patients

ObjectiveFine needle cytology (FNC) is the first‐line diagnostic method to determine the benign or malignant nature of thyroid nodules. The gray zone of cytological classifications remains, however, a crucial and challenging area for cytopathologists.Design, patients and measurementsIn the present study, 141 thyroid cytological samples, with ultrasonographic suspicious features, have been prospectively analysed. Molecular analyses were performed by an innovative technology using two multiplex PCRs for the amplification of BRAF, N‐H‐K‐RAS and RET exon genes. RNA samples were studied for RET/PTC1 and RET/PTC3 rearrangements by PCR amplification, and the conditions were set‐up to study, with a single experiment, both wild‐type PAX8 and PAX8/PPARɣ rearrangements. In total, 111 samples were examined for BRAF, N‐H‐KRAS and RET genes. An ultrasonographic, cytological and molecular correlation was also carried out in an attempt to suggest a possible way to manage the patients with thyroid nodules. Cyto‐histological correlation was available in 115 cases, and it was used to verify the global diagnostic accuracy of this combined approach.ResultsAccording to the histopathological diagnosis, FNC accuracy was 100% for TIR5 and metastases; 89% for TIR4; 84% for TIR3A and 58% for TIR3B. About 11% of the studied samples showed either RET‐PTC1 or RET/PTC3 chromosomal rearrangements, and only one sample simultaneously presented RET/PTC1 and RET/PTC3 rearrangements. PAX8/PPARɣ rearrangement was found in 6% of the samples.ConclusionsA multidisciplinary approach to the thyroid is therefore necessary to develop innovative methods suitable for an improved diagnostic and prognostic definition of thyroid cancer.

EP1.04-11 Frequency of Microsatellite Instability (MSI) in Brazilian TKI Non-Treatable Non-Small Cell Lung Cancer (NSCLC) Patients

Lung cancer is the most common cause of cancer deaths worldwide. Personalized medicine based on driver mutations has improved tailored treatment for patients with non-small cell lung cancer (NSCLC). However, only a subset of patients is benefited with tyrosine kinase inhibitors. Non-treatable advanced NSCLC patients may be eligible for treatment with immunotherapies. Recently, microsatellite instability (MSI) phenotype was reported as a predictive biomarker of response to immunotherapy, making it a crucial biomarker for clinical management for NSCLC patients. This study aimed to determine MSI frequencies in Brazilian NSCLC patients who are not eligible for TKi therapy. 522 patients diagnosed with NSCLC patients at Barretos Cancer Hospital (Brazil) were evaluated. All samples were molecularly analyzed for EGFR (exons 18, 19, 20 and 21), KRAS (codons 12/13) and BRAF (exon 11 and 15) mutations by PCR followed by Sanger direct sequencing. The molecular MSI evaluation was performed using a hexa-plex marker panel by PCR followed by fragment analysis. The mean age of the patients was 61y (22-87) and 54% were male. EGFR, KRAS, and BRAF wild-type status were identified in 62.6% (327/522) of cases. In 297 EGFR/KRAS/BRAF wild-type cases, MSI analysis was performed and we observed the presence of MSI-H in only three (1%) cases. Overall, two out of these three patients were female and one was male, the age at the diagnosis ranged from 55 to 76 years old, they were current or former smokers and all three cases were diagnosed at stage IV. Of note, one of the patients with MSI-high status received treatment with immune checkpoint inhibitor, and he is the only one out of the three patients that remains alive with 51-months survival. The frequency of MSI-high status is low in Brazilian NSCLC patients, in accordance with other population literature. Nevertheless, these MSI-positive patients are eligible for immunotherapy approaches.

Acral Lentiginous Melanomas Harbour Intratumor Heterogeneity in BRAF Exon 15, With Mutations Distinct From V600E/V600K

The choice of appropriate therapeutic strategies may be influenced by intratumor heterogeneity and makes cancer treatment considerably more challenging. We aimed to evaluate the heterogeneity of BRAF exon 15 mutations in different areas of acral lentiginous melanoma (ALM). The entire exon 15 was sequenced in 4 different areas of paraffin-embedded samples from 26 patients with ALM. A total of 26 of 49 cases of ≥1 mm in depth of ALM identified by clinical, anatomical, and pathological data fulfilled the inclusion and exclusion criteria for this study. Tumors had a mean Breslow depth of 7.2 mm and an average mitotic index of 3 mitosis/mm. Mutations distinct from the common V600E and V600K were detected in 31%, and intratumor heterogeneity was observed in 31% of samples. Interestingly, 63.5% of all mutations had been previously associated with cancer. Most (62.5%) of the missense BRAF exon 15 mutations found in the ALM samples examined here were deemed "detrimental" for protein function according to at least 2 functional prediction programs, and 3 mutations (37.5%) were predicted to be "neutral," with no effect on protein function. BRAF exon 15 mutations were detected frequently in ALM and displayed heterogeneity, a finding to be further investigated.

P14.110 Primary leptomeningeal melanocytic tumors - clinical course and molecular pathological aspects of an underestimated entity

Abstract BACKGROUND Primary leptomeningeal melanocytic tumors (PLMT) of the central nervous system are extremely rare, usually benign pigmented tumors. The aim of our study is to present two complex cases of PLMT with discussion of clinical and molecular aspects. MATERIAL AND METHODS Medical charts of two patients from our institution with PLMT were reviewed. Oncogenic common gene mutations in GNAQ (Q209, exon 5), GNA11 (Q209, exon 5), TERT promoter and BRAF (exon 11, 15) were analyzed and a genome-wide DNA methylation array (Infinium Human-MethylationEPIC BeadChip) was performed. Brain tumors were categorized according to their DNA methylation profile using the brain tumor classifier algorithm (https://www.molecularneuropathology.org/mnp). RESULTS Case1: A 15 year-old boy presented with a large tumor in the in the cerebellopontine angle with extension to the cavernous sinus and the middle fossa in June 2013. Consequently, partial resection of the PLMT was performed followed by photon radiation therapy. First recurrence developed after 12 months. Despite reradiation with proton therapy, reresection, and several chemotherapies the patient died 4 years and 3 month after initial diagnosis. Histopathological examination of the resected tumor sample revealed a pigmented neoplasm with epithelioid cells with strong expression of melanocytic markers Melan A and HMB 45. In the recurrent biopsy samples, an increase in the Ki-67/MIB index from 2 to 10 % was observed. Molecular analysis showed a characteristic GNA11 Q209L mutation that supports the diagnosis of PLMT. Case2: In April 2016 the resection of a PLMT in a 42 year-old female in the pinealis region was performed. First recurrence developed after 21 months and reresection followed by proton therapy was performed. A distant tumor developed 7 month later and after resection the patient received local proton therapy. Only 1 month later a new tentorial tumor was detected and treated by proton therapy. Tumor tissue of the local recurrence showed focal epitheloid cell morphology with brain invasion, tumor necrosis and increased mitotic activity. KI-67/MIB1 index was approximately 3% in the first and above 10 % in the second and third operation. The hotspot mutation in GNAQ Q209L was found in all tumor samples. In addition to histological signs of malignancy, an increase in chromosomal aberrations was seen at recurrence as a sign of malignant progression. To-date the patient is alive but new cranial metastases were detected. CONCLUSION Though PLMT is defined as benign lesion, the treatment is complicated by early local recurrence as well as intracranial and spinal metastases. For the first time malignant transformation during the clinical course of PLMT was confirmed by histology and DNA methylation array.

Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma.

Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was identified by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers.

An unusual diagnosis of a dural based intracranial lesion

An unusual diagnosis of a dural based intracranial lesion

NRAS Q61R and BRAF G466A mutations in atypical melanocytic lesions newly arising in advanced melanoma patients treated with vemurafenib.

BRAF inhibition has improved overall survival in patients with BRAF mutant melanoma, but this is associated with a range of known and predictable cutaneous side effects, including squamous cell carcinomas associated with RAS mutations. We identified three severely dysplastic nevi, one atypical intraepidermal melanocytic proliferation, and four melanoma in situ lesions, newly arising in four patients undergoing treatment with vemurafenib. To characterize mutations in these atypical melanocytic lesions, we used a custom iPlex panel detecting 74 mutations in 13 genes known to play a role in melanoma pathogenesis. We identified an NRAS mutation at codon 61 (Q61R) and a rare BRAF exon 11 mutation (G466A) in atypical melanocytic lesions that arose in patients treated with vemurafenib. There appears to be development or accelerated growth of atypical melanocytic lesions in the setting of BRAF inhibition. Our results underscore the need for careful surveillance for melanocytic lesions in patients on BRAF inhibitor therapy and shed light on potential mechanisms for melanoma pathogenesis in the context of BRAF pathway blockade. Further studies are warranted to show a causal relationship.

KRAS, NRAS and BRAF Mutations Are Highly Enriched in TRI12 Chronic Lymphocytic Leukemia and Are Associated to Shorter Time to First Treatment

BackgroundExtensive sequencing data highlighted the recurrence of mutations resulting in constitutive MAP kinase signaling in chronic lymphocytic leukemia (CLL), and proposed these mutations as late drivers of CLL progression (X.S. Puente, Nature, 2015; D.A. Landau, Nature, 2015). Although some preliminary studies suggested KRAS, NRAS and BRAF mutations as possibly associated with trisomy 12 (TRI12) and/or unmutated (UM) IGHV genes (C.D. Herling, Blood, 2016; K. Takahashi, Blood, 2018), dedicated analysis of these aspects is still missing.AimTo correlate the occurrence of KRAS, NRAS and BRAF mutations with specific clinico-biological features in CLL.MethodsThe study included 534 primary CLL purposely enriched with 300 TRI12 cases: 190 cases with TRI12 as sole chromosomal aberration (TRI12only) and 110 cases harboring TRI12 plus other chromosomal aberrations (TRI12plus). The cohort was also enriched in cases with NOTCH1 aberrations (n=214) to evaluate their possible role in RAS-RAF mutations incidence.Amplicon-based targeted next-generation sequencing assay was performed using Miseq Illumina on DNA from purified CLL samples. Analyzed amplicons mapped in the hotspot regions of KRAS, NRAS (exons 2, 3 and 4) and BRAF (exons 11 and 15) genes. Variant allele frequency (VAF) was obtained by Miseq report. CLL cases were characterized for IGHV mutational status, main cytogenetic abnormalities, NOTCH1 and TP53 aberrations. Time to first treatment (TTFT) data were correlated with molecular findings.ResultsWe found 90 missense point mutations (Fig.1) in 64 CLL cases, with prevalence of KRAS (44 mutations in 38 patients), followed by BRAF (31 mutations in 24 patients) and NRAS (15 mutations in 13 patients). Nearly all mutations have been previously associated with gain-of-function phenotype and increased RAS/ERK downstream signaling. The co-occurrence of 2 mutated genes (mainly KRAS and BRAF) were observed in 11 patients. Mutations were mainly subclonal (median VAF 6.15%, range 1.3%-61.6%) with only one third of mutations (27/90) above 15% VAF.A high association between the presence of KRAS/NRAS/BRAF mutations and UM IGHV and the presence of TRI12 was observed. Overall, 87.3% of KRAS/NRAS/BRAF mutated cases had a UM IGHV (p<0.0001) and 79.7% were TRI12 CLL (p<0.0001) (Fig.2). Moreover, in the context of the UM IGHV CLL group (332 cases), TRI12 CLL had a 4-fold higher KRAS/NRAS/BRAF mutation frequency (46/182, 25.3%) than non-TRI12 (9/150, 6%; p<0.0001). Indeed, we found 29/182 (15.9%) KRAS, 9/182 (4.9%) NRAS and 17/182 (9.3%) BRAF mutated cases in the TRI12 group vs. 4/150 (2.7%) KRAS, 3/150 (2%) NRAS and 3/150 (2%) BRAF mutated cases in the non-TRI12 group. The KRAS/NRAS/BRAF mutation frequency was even higher when considering the TRI12only group in the context of UM IGHV (38/133, 28.6%). Conversely, CLL cases with del(13q) as sole chromosomal aberration (n=94) showed the lowest frequency of KRAS/NRAS/BRAF mutations (2/94, 2.1% in the whole cohort and 2/53, 3.8% in the context of UM IGHV cases).Furthermore, a higher prevalence of KRAS/NRAS/BRAF mutations was found in NOTCH1 wild type (wt) (22.1%) when compared to NOTCH1 mutated (11.2%) cases, in the context of the UM IGHV group (p=0.008), pointing to a mutual exclusivity of these mutations in the pathogenesis of disease.No other significant associations with clinical variables as RAI stage at diagnosis, presence of TP53 mutations or gender were observed.We finally evaluated if KRAS/NRAS/BRAF mutations had an impact on TTFT in the context of UM IGHV/TRI12only/NOTCH1wt, that showed the highest incidence of mutations (26/70, 37%). Both KRAS mutations alone (p=0.005) and KRAS/NRAS mutations (p=0.04) were associated with shorter TTFT (Fig.3).ConclusionsTaken together our data show that KRAS, NRAS and BRAF mutations are almost exclusively found in UM IGHV/TRI12/NOTCH1wt CLL and, in the context of this CLL group the presence of KRAS and NRAS mutations is associated with unfavorable prognosis.The high incidence of KRAS, NRAS and BRAF activating mutations in TRI12 CLL together with the observation of higher level of ERK phosphorylation (S. Decker, Blood, 2012) and higher sensitivity to MEK/ERK inhibitors (S. Dietrich, J Clin Invest, 2018) described in the TRI12, reinforce the evidence of an essential role for MEK/ERK signaling in TRI12 CLL, pointing to this pathway as the most appropriate therapeutic target. [Display omitted] DisclosuresZaja:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma.

To determine the role of BRAFV600E mutation and MAPK signaling as well as the effects of BRAF and MEK directed therapy in gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN), with a focus on highly aggressive gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Using Sanger sequencing of BRAF exon 15 we determined the frequency of BRAFV600E mutations in 71 primary GEP-NENs. MEK phosphorylation was examined by immunohistochemistry in corresponding tissue samples. To evaluate the biological relevance of BRAFV600E mutation and MAPK signaling in GEP-NECs, effects of a pharmacological BRAF and MEK inhibition were analyzed in NEC cell lines both in vitro and in vivo. BRAFV600E mutation was detected in 9.9% of all GEP-NENs. Interestingly, only NECs of the colon harbored BRAFV600E mutations, leading to a mutation frequency of 46.7% in this subgroup of patients. In addition, a BRAFV600E mutation was significantly associated with high levels of MEK phosphorylation (pMEK) and advanced tumor stages. Pharmacological inhibition of BRAF and MEK abrogated NEC cell growth, inducing G1 cell cycle arrest and apoptosis only in BRAFV600E mutated cells. BRAF inhibitor dabrafenib and MEK inhibitor trametinib prevented growth of BRAFV600E positive NEC xenografts. High frequencies of BRAFV600E mutation and elevated expression levels of pMEK were detected in biologically aggressive and highly proliferative colorectal NECs. We provide evidence that targeting BRAF oncogene may represent a therapeutic strategy for patients with BRAF mutant colorectal NECs.

Mutation Status and Immunohistochemical Correlation of KRAS, NRAS, and BRAF in 260 Chinese Colorectal and Gastric Cancers.

KRAS, NRAS and BRAF are kinases involved in the RAS-RAF-MAPK signaling pathway and also potential tumor-driven genes. Patients with KRAS/NRAS/BRAF mutations are resistant to anti-EGFR monoclonal antibody therapy. The main purpose of this study is to investigate the mutation status and distribution of KRAS/NRAS/BRAF in Chinese colorectal and gastric cancers, and to explore the histopathological changes and related immunohistochemical marker changes caused by these mutations. The mutation status of KRAS (exons 2, codon 12/13), NRAS (exons 2/3/4, codon 12/13/59/61/117/146) and BRAF (exons 15, codon 600) were detected by amplification refractory mutation system polymerase chain reaction (ARMS-PCR) in 86 colon cancer, 140 rectal cancer and 34 gastric cancer tissues. Then, the frequencies and distribution of KRAS/NRAS/BRAF mutations were described in detail. Furthermore, the relationship between KRAS/NRAS/BRAF mutations and the features of histopathological and related immunohistochemical markers were analyzed. The results showed that KRAS/NRAS/BRAF mutation rates in colon cancer were 44.2, 1.2, and 3.5%; in rectal cancer were 37.1, 4.3, and 0.7%; in gastric cancer were none, none and 2.9%. The mutation rate of KRAS in female (48.8%) is significantly higher than that of male (27.8%), and the mutation rate increased with the higher degree of differentiation. Additionally, the mutation rate of BRAF detected by ARMS-PCR (1.77%) was significantly lower than that by immunohistochemistry (4.11%). It also showed that the KRAS/NRAS/BRAF mutation status had a certain relationship with the expression of some immunohistochemical markers. This study provides more data support for clinical research on KRAS/NRAS/BRAF mutation in CRCs or gastric cancers.

Success of genomic profiling of non-small cell lung cancer biopsies obtained by trans-thoracic percutaneous needle biopsy.

Genomic profiling for personalized targeted therapy has become standard of care. We report the success of genomic profiling of non-small cell lung cancer (NSCLC) obtained by trans-thoracic needle biopsy (TTNB) in a single center experience. Patients with NSCLC who underwent TTNB for genomic were identified. Pathology specimens were evaluated for tumor adequacy and then analyzed for selected exons of epidermal growth factor receptor, KRAS, BRAF, PIK3CA, and ERBB2. ALK rearrangements were detected with fluorescence in situ hybridization and/or immunohistochemistry. Technical success was recorded and the factors affecting successful profiling were evaluated. Complications (pneumothorax, hemorrhage, and admission) were recorded. Comparison of yield and complications were done between the two groups (core biopsy and fine needle aspiration only group). Utility of PET-CT to guide the needle track for optimized yield was assessed in a subset of patients. Between December 6, 2009, and December 30, 2016, 765 patients with NSCLC underwent TTNB. Five-hundred and seventy-seven of 765 (75%) of all TTNB were profiled, for genomic analysis. Five-hundred and eight of 577 (88%) were successfully profiled. The number of samples obtained ranged from 1 to 10 (1 to 2 cm,18 to 20 G). Lesions biopsied ranged in size from 0.6 to 16 cm. No statistically significant difference was observed in the incidence of pneumothorax between two groups (P = 0.26). PET guidance was not found to be statistically significant ( P = 0.79) in the overall yield. Computed tomographic guided TTNB is a safe and efficacious technique for genomic profiling, enables the acquisition of sufficient tissue for genetic mutation analyses allowing for personalized therapy with an acceptable complication rate.

BRAF fusions identified in melanomas have variable treatment responses and phenotypes.

Oncogenic BRAF fusions have emerged as an alternate mechanism for BRAF activation in melanomas and other cancers. A number of BRAF fusions with different 5' gene partners and BRAF exon breakpoints have been described, but the effects of different partners and breakpoints on cancer phenotypes and treatment responses has not been well characterized. Targeted RNA sequencing was used to screen 60 melanoma patient-derived xenograft (PDX) models for BRAF fusions. We identified three unique BRAF fusions, including a novel SEPT3-BRAF fusion, occurring in four tumors (4/60, 6.7%), all of which were "pan-negative" (lacking other common mutations) (4/18, 22.2%). The BRAF fusion PDX models showed variable growth rates and responses to MAPK inhibitors in vivo. Overexpression of BRAF fusions identified in our study, as well as other BRAF fusions previously identified in melanomas, resulted in a high degree of variability in 2D proliferation and 3D invasion between the different fusions. While exogenously expressed BRAF fusions all responded to MAPK inhibition in vitro, we observed potential differences in signaling and feedback mechanisms. In summary, BRAF fusions are actionable therapeutic targets, however there are significant differences in phenotypes, treatment responses, and signaling which may be clinically relevant.

Targeted Tissue and Cell-Free Tumor DNA Sequencing of Advanced Lung Squamous-Cell Carcinoma Reveals Clinically Significant Prevalence of Actionable Alterations

BackgroundMajor guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cell-free circulating tumor DNA, facilitates reevaluation of this premise. Patients and MethodsWe retrospectively evaluated the prevalence of actionable alterations in 2 distinct LUSC cohorts totaling 492 patients. A total of 410 consecutive patients with stage 3B or 4 LUSC were tested with a targeted cell-free circulating DNA NGS assay, and 82 patients with LUSC of any stage were tested with a tissue NGS cancer panel. ResultsIn the overall cohort, 467 patients (94.9%) had a diagnosis of LUSC, and 25 patients (5.1%) had mixed histology with a squamous component. A total of 10.5% of the LUSC subgroup had somatic alterations with therapeutic relevance, including in EGFR (2.8%), ALK/ROS1 (1.3%), BRAF (1.5%), and MET amplification or exon 14 skipping (5.1%). Sixteen percent of patients with mixed histology had an actionable alteration. In the LUSC subgroup, 3 evaluable patients were treated with targeted therapy for an actionable alteration; all of them experienced partial response. ConclusionIn this large, real-world LUSC cohort, we observed a clinically significant prevalence of actionable alterations. Accurate local histopathologic assessment in advanced-stage LUSC can be challenging. Further evaluation of the genomic landscape in this setting is warranted to potentially identify underappreciated treatment options.

Abstract 3697: High PD-L1 expression and targetable mutations occur independently in NSCLC and are not influenced by smoking or race

Abstract Background: Targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs), and immunotherapy (IO) with PD-L1 inhibitors have revolutionized first-line treatment options for advanced non-small cell lung cancer (NSCLC). Several small studies suggest that high PD-L1 expression rarely overlaps with classic driver mutations, yet it remains unclear how smoking history, race, and stage may influence the interaction between PD-L1 expression and targetable driver mutations. In our racially diverse population with a high proportion of African American patients, we investigated whether high PD-L1 expression occurs concurrently with targetable mutations in EGFR, BRAF, and MET and whether smoking history, stage, or race alters this distribution. Methods: In this retrospective study, we selected all NSCLC patients of any stage at our institution whose tumor samples had undergone both in-house next-generation sequencing (NGS) and PD-L1 staining with the Dako Clone 28.8 antibody between March 2016 to July 2017. Our validated NGS assay, the OncoPlus, detects mutations, deletions, and insertions in 147 clinically reported genes, including all classic driver mutations in lung cancer. Patient demographics, treatments and response data were extracted from the electronic medical record through October 1, 2017. We then used Wilson proportion test and chi-squared analysis to analyze whether PD-L1 expression and actionable mutations varied by race, smoking, age and stage. Results: 114 cases had both PD-L1 staining and NGS analysis available. 71 (62.2%) patients had ≥10 pack year smoking history while 21 (18.4%) patients were never smokers. 41 (35.9%) patients were African American, 62 (54.4%) Caucasian and 7 (6.1%) were Asian. 26 (22.8%) had high expression of PD-L1 (≥50% cells staining positive), and 15 (13.2%), 1 (0.8%), 4 (3.5%), and 6 (5.2%) patients, respectively, had an EGFR, BRAF (V600E), ERBB2, and MET exon 14 skip mutation. 4/26 patients with high PD-L1 expression had a targetable mutation, compared to 22/88 patients with low PD-L1 expression (p&amp;lt;0.001, Wilson test). This distribution did not vary significantly when stratified by race, stage, or smoking history. Conclusions: We present here our institution's novel workflow of obtaining both in-house NGS and PD-L1 staining at time of initial biopsy, in a diverse population with 1/3 African American patients. We demonstrate that high PD-L1 expression and actionable driver mutations infrequently coexist in the same tumor, and this relationship is not driven by smoking, race, or stage. Our results strongly suggest the necessity of obtaining both PD-L1 staining and NGS at time of initial biopsy to identify optimal therapies for patients with advanced disease. Our results imply that patients with actionable driver mutations are unlikely to benefit from later treatment with IO, which we hope to test explicitly in future studies. Citation Format: Noura J. Choudhury, Mansooreh Eghtesad, John Cursio, Sabah Kadri, Lauren Ritterhouse, Jeremy Segal, Aliya Husain, Jyoti D. Patel. High PD-L1 expression and targetable mutations occur independently in NSCLC and are not influenced by smoking or race [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 3697.

Cetuximab Alone or With Irinotecan for Resistant KRAS-, NRAS-, BRAF- and PIK3CA-wild-type Metastatic Colorectal Cancer: The AGITG Randomized Phase II ICECREAM Study

BackgroundThe Irinotecan Cetuximab Evaluation and Cetuximab Response Evaluation (ICECREAM) study assessed the efficacy of cetuximab monotherapy compared with cetuximab combined with chemotherapy for quadruple wild-type (KRAS, NRAS, BRAF, or P13KCA exon 20) metastatic colorectal cancer. Patients and MethodsPatients were enrolled in an open-label, multicenter, phase II trial and randomly assigned to cetuximab 400 mg/m2, then 250 mg/m2 cetuximab weekly, with or without irinotecan 180 mg/m2 every 2 weeks. The primary endpoint was 6-month progression-free survival; secondary endpoints were response rate, overall survival, toxicity, and quality of life. ResultsFrom 2012 to 2016, 48 patients were recruited. Two were ineligible, and 2 were not evaluable for response. Characteristics were balanced, except gender (male, 62% vs. 72%) and primary sidedness (left, 95% vs. 68%). For cetuximab compared with cetuximab-irinotecan, progression-free survival was 14% versus 41% (hazard ratio, 0.39; 95% confidence interval, 0.20-0.78; P = .008); response rate was 10% (2 partial responses) versus 38% (1 complete, 8 partial); P = .04. Grade 3 to 4 toxicities were less with cetuximab monotherapy (23% vs. 50%); global and specific quality of life scores did not differ. ConclusionIn comparison with cetuximab alone, cetuximab plus irinotecan increases the response rate and delays progression in irinotecan-resistant RAS wild-type colorectal cancer. This echoes data from molecularly unselected patients.

Combined assessment of the TNM stage and BRAF mutational status at diagnosis in sporadic colorectal cancer patients.

The prognostic impact of KRAS mutations and other KRAS-related and non-related genes such as BRAF, NRAS and TP53, on sporadic colorectal cancer (sCRC) remain controversial and/or have not been fully established. Here we investigated the frequency of such mutations in primary sCRC tumors and their impact on patient progression-free survival (PFS) and overall survival (OS). Primary tumor tissues from 87 sCRC patients were analysed using a custom-built next generation sequencing (NGS) panel to assess the hotspot mutated regions of KRAS/NRAS (exons 2, 3 and 4), BRAF (exon 15) and TP53 (all exons). Overall, mutations in these genes were detected in 46/87 sCRC tumors analyzed (53%) with the following frequencies per gene: TP53, 33%; KRAS, 28%; BRAF, 7%; and NRAS, 1%. A significant association was found between KRAS mutations and right side colon tumor location (p=0.05), well-differentiated tumors (p=0.04) and absence of lymphovascular invasion (p=0.05). In turn, BRAF-mutated tumors frequently corresponded to poorly- or moderately-differentiated sCRC (p=0.02) and showed a higher frequency of peritoneal carcinomatosis (p=0.006) and microsatellite instability (p=0.007). From the prognostic point of view, the BRAF mutational status together with the TNM stage were the only variables that showed an independent adverse impact on patient outcome in the multivariate analyses for both PFS and OS. Based on these results a scoring system was built and patients were classified into three prognostic subgroups with different PFS rates at 2 years: 91% vs. 77% vs. 0%, respectively (p<0.0001). Additional prospective studies in larger series of sCRC patients where mutations in genes other than those investigated here are required to validate the utility of the proposed predictive model.