Wild-type BRAF Research Articles

Multiple lectin assays in detecting glycol-alteration status of serum NRG1 in papillary thyroid cancer.

BackgroundNeuregulin 1 (NRG1) is a membrane glycoprotein mediating cell-to-cell signaling and has a crucial role in the growth and development of various organ systems. Our study explored its diagnostic value in distinguishing BRAF V600E mutant status in papillary thyroid cancer (PTC) patients by analyzing multiple glycan patterns of serum NRG1 through lectin assays.MethodsWe first extracted serum from PTC patients and tested BRAF V600E mutation by immunohistochemical (IHC) staining. Then we applied antibody overlay lectin microarray and lectin blot to detect glycol-alterations of NRG1. Then Aleuria aurantia lectin (AAL) ELISA was performed according to ELISA index to test the protein fucosylation level of NRG1 (Fuc-NRG1).ResultsWe got glycan profiles of 14 lectins, including GNL, GSL2, AAL, BPL, ECL, CAL, NML, HHL, PHA-L, RCA-I, ConA, DBA, PWA and LEL. Six of them, namely, GSL2, BPL, NML, HHL, PHA-L and LEL, had significantly increased binding affinity capacity in BRAF(+) PTC compared with BRAF(-) PTC controls. LEL, BPL and NML tended to bind to NRG1 in BRAF(+) PTC group. Both AAL ELISA and protein ELISA assays showed that the fucosylated structures of NRG1 had a remarkable increase in BRAF V600E mutant PTC patients compared with BRAF wild type PTC controls.ConclusionsThis study sheds a new light on the role of NRG1 glycosylation in PTC. NRG1 could serve as a supplementary glycobiomarker for BRAF indicator in discrimination of PTC patients with BRAF wild type negative fine needle aspiration results.

Abstract 2302: The mechanism of activation of monomeric B-Raf V600E

Abstract B-Raf V600E mutations are frequent in cancer, including over 50% of all melanoma. B-Raf, like many other kinases, undergoes structural changes as it transitions between the inactive and active conformations. Key among these changes are the movement of αC helix from an inactive “OUT” conformation to an active “IN” conformation, and the movement of the activation loop from a closed inactive conformation to an open active conformation. Yet even with the knowledge of what the inactive and active conformations are, questions remain concerning how the V600E mutation activates B-Raf. In particular, we seek to understand why B-Raf V600E is constitutively active in its monomeric state while wild type B-Raf requires dimerization. Here, we investigate the differences between the wild type and mutant B-Raf monomers using molecular dynamics (MD) simulations. We included ATP in the nucleotide binding pocket to more fully explore how it is positioned within B-Raf. Our results show that the 1 µs simulations are too short to capture large conformational changes, however some of the simulations show a transient formation and breaking of the key salt-bridge between Lys483 and Glu501, which gives insight into the movement of the αC helix between the OUT and IN conformations. In addition, the dihedral angles around the DFG motif move towards a more active configuration for B-Raf V600E, while those of the wild type adopt a more inactive orientation. These simulations aid in our understanding of the dynamics of activation in B-Raf V600E. Citation Format: Ryan C. Maloney, Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov. The mechanism of activation of monomeric B-Raf V600E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2302.

Abstract 1481: DAY101, a potent pan-RAF inhibitor with activity in preclinical models harboring MAPK pathway alterations beyond BRAF V600E mutation

Abstract DAY101 (formerly TAK-580) is an oral, CNS-penetrant, selective small molecule pan-RAF kinase inhibitor that is equipotent against BRAF V600E, wildtype BRAF, and CRAF and can inhibit both RAF monomers and dimers, including BRAF fusions. KIAA1549:BRAF fusions have been identified as a key oncogenic driver for pilocytic astrocytoma (Jones et al. 2008). DAY101 has demonstrated significant activity against this fusion preclinically (Sun et al. 2017). Clinical data for DAY101 has shown a durable 63% response rate with good tolerability in children with low-grade glioma (LGG) harboring RAF fusions (Wright et al. 2020). To explore the potential impact of DAY101 in adult solid tumors harboring specific genomic alterations beyond BRAF V600E, a panel of tumor cell lines and PDX models harboring BRAF fusions, non V600 BRAF mutations or RAS mutations were assessed for anti-proliferative activity in response to DAY101. Furthermore, hypothesis-driven MAPK pathway node inhibitor combinations were explored for potential synergy. While performing favourably in preclinical in vivo studies, DAY101 does exhibit some technical challenges in long-term in vitro assays, particularly in those that extend for more than three days. Accordingly, the tool compound TAK-632, which exhibits comparable biochemical and cellular potency to DAY101, was used alternatively as needed for in vitro assays. Single-agent anti-proliferative activity was observed in a melanoma BRAF fusion PDX model treated with TAK-632 ex vivo, with less sensitivity observed in models beyond melanoma harboring other BRAF fusions. Ongoing combination studies with MEK inhibitors demonstrated strong synergy in PDX models harboring BRAF fusions ex vivo, as assessed by Bliss independence or other relevant scoring systems. In cell lines or PDX models harboring non V600 BRAF mutations, in vitro combination studies demonstrated strong synergy when DAY101 was combined with MEK inhibitors, as assessed by Bliss independence score. DAY101 exhibited modest single agent anti-proliferative activity in a panel of KRAS mutant cell lines in vitro and ongoing combination studies will assess potential synergy when combined with MAPK pathway node inhibitors. DAY101 is currently in clinical development for the treatment of patients under 25 years old with LGG harboring RAF alterations, including BRAF fusions and BRAF mutations. Inhibition of both RAF monomers and dimers by DAY101 enables a broader indication selection strategy, beyond LGG and BRAF alterations. Ongoing translational work will highlight the potential utility of DAY101 in adult tumors harboring RAS or RAF alterations, both as a single agent and in hypothesis-driven combinations. Citation Format: Eleni Venetsanakos, Mike Preigh, Jeannie Hou, Michael C. Cox, Jeremy Bender, Sam C. Blackman. DAY101, a potent pan-RAF inhibitor with activity in preclinical models harboring MAPK pathway alterations beyond BRAF V600E mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1481.

Abstract 1263: Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS-G12C inhibitors through a vertical pathway inhibition strategy

Abstract KRAS is one of the most frequently mutated oncogenes in cancer, stimulating tumor growth through activation of the RAS/RAF/MEK/ERK pathway. G12C inhibitors (G12Ci), sotorasib (AMG 510) and adagrasib (MTRX849), have demonstrated promising antitumor activity in patients with KRAS G12C mutant (mt) non-small cell lung cancer (NSCLC). Several recent studies have shown that simultaneous targeting of multiple nodes in the RAS/RAF/MEK/ERK pathway may be optimal for durable pathway inhibition and response. VS-6766 is a unique dual RAF/MEK inhibitor. In contrast to other MEK inhibitors, VS-6766 is a potent allosteric inhibitor of MEK kinase activity which promotes a dominant negative RAF/MEK complex preventing phosphorylation of MEK by wildtype BRAF, BRAF V600E mt and CRAF. This unique mechanism allows VS-6766 to block MEK signaling without the compensatory activation of MEK that appears to limit the efficacy of other inhibitors. Clinical studies are ongoing evaluating VS-6766 alone or in combination with the focal adhesion kinase (FAK) inhibitor defactinib for the treatment of various solid tumors. Here, we tested the hypothesis that vertical pharmacological blockade of RAS, RAF and MEK with G12Ci in combination with VS-6766 might yield superior pathway blockade and antitumor efficacy. In 3D proliferation assays in vitro, VS-6766 was synergistic with both sotorasib and adagrasib in reducing viability of a panel of KRAS G12C mt NSCLC and colorectal cancer cell lines. Accordingly, VS-6766 effectively suppressed RAS pathway signaling (pMEK, pERK, p-p90RSK) across KRAS G12C mt NSCLC cell lines as a single agent, and the combination of VS-6766 + G12Ci showed improved depth and duration of RAS pathway signaling relative to G12Ci alone. Similarly, in the H2122 KRAS G12C mt NSCLC xenograft model, VS-6766 combination with sotorasib showed improved inhibition of pMEK and pERK in tumors relative to sotorasib alone. In KRAS G12C mt NSCLC xenograft models in vivo, combination with VS-6766 (0.3 mg/kg QD) augmented tumor growth inhibition by sotorasib (30 mg/kg QD) in the H2122 KRAS G12C mt NSCLC model, whereas trametinib (0.3 mg/kg QD) was much less effective in augmenting sotorasib efficacy. Sotorasib monotherapy induced >20% tumor regression in 0/10 mice, whereas combination of VS-6766, trametinib or a FAK inhibitor with sotorasib induced >20% tumor regression in 5/10, 1/10 and 4/10 mice, respectively, following 10 days of treatment. Strikingly, triple combination of VS-6766, sotorasib and a FAK inhibitor conferred tumor reductions of ≥35% in 10/10 mice. Similarly, in the H358 KRAS G12C mt NSCLC model, triple combination of VS-6766, sotorasib and a FAK inhibitor induced >35% tumor regression in 10/10 mice following 21 days of treatment. These results support the clinical evaluation of VS-6766 ± defactinib in combination with a G12C inhibitor for treatment of KRAS G12C mt solid tumors. Citation Format: Silvia Coma, Sanjib Chowdhury, Jonathan A. Pachter. Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS-G12C inhibitors through a vertical pathway inhibition strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1263.

Abstract 579: Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis

Abstract Background Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. Methods A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. Results The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF and, eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. G12D, G12V and G12R-mutant KRAS alleles were detected with FAM-labeled probes, G13D-mutant KRAS allele and wildtype KRAS allele were detected with HEX-labeled probes, G12S and G12C-mutant KRAS alleles were detected with Cy5-labeled probes, and V600E-mutant BRAF allele and wildtype BRAF allele were detected with CAL Fluor Red 610-labeled probes. The results showed that the peaks of the Tm distributions of the DNA groups were separated by 3.5 °C on average, while the standard deviation of the Tm distributions was 0.23 °C on average. The difference in Tm was 15 times larger than the standard deviation, enabling more accurate genotyping than when using the fluorescence intensity. Conclusion We have proposed a measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. We plan to evaluate the effect of this method on measurement using clinical specimens of pancreatic cancer patients in the future. Citation Format: Junko Tanaka, Tatsuo Nakagawa, Takeshi Ishida, Yoshio Kamura, Akiko Shiratori, Kunio Harada, Chihiro Uematsu. Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 579.

Abstract 78: A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors

Abstract BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical RAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric RAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. Here we identify and characterize a third class of RAF inhibitors that preferentially binds and inhibits dimeric over monomeric BRAF(V600E). Using long-timescale molecular dynamics (MD) simulations, we observed distinct dynamic features of RAF inhibitors bound to monomeric and dimeric BRAF, thus providing a structural explanation for the selectivity of these drugs. Biochemical analysis using RAF dimer selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of RAF monomer- plus RAF dimer selective inhibitor plus a MEK inhibitor that potently disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical RAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the RAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher therapeutic index. Finally, off-label use of the triple combination in a patient with a stage IV BRAF(V600E) colorectal cancer that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective RAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Maxwell R. Tucker, Peter M. Ung, Min Xiao, Zoi Karoulia, Xuewei Wu, Stuart A. Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Qi Wang, David E. Shaw, Poulikos I. Poulikakos. A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 78.

Predicting BRAF V600E mutation in glioblastoma: utility of radiographic features

Detection of BRAF V600E mutation in glioblastomas (GBMs) is important because of potential therapeutic implications. Still, the relative paucity of these mutations makes molecular detection in all GBMs controversial. In the present study, we analyzed clinical, radiographic and pathologic features of 12 BRAF V600E-mutant GBMs and 12 matched controls from 2 institutions. We found that a majority of BRAF V600E-mutant GBMs displayed a combination of well-circumscribed lesions, large cystic components with thin walls and solid cortical component on MRI, but with some overlap with matched BRAF wildtype controls (p = 0.069). BRAF V600E-mutant GBMs were also apt to gross total resection (83% vs 17%, p = 0.016) and morphologically displayed epithelioid features (83% vs 0%, p < 0.0001). Identification of these clinical, radiographic, and pathologic characteristics should prompt testing for BRAF V600E in IDH-wildtype GBM.

Immune Checkpoint Inhibitors Restore Function of Lesion Infiltrating T Lymphocytes in Langerhans Cell Histiocytosis

Langerhans Cell Histiocytosis (LCH) is characterized by inflammatory lesions with pathologic CD207+ dendritic cells (DCs) and constitutively activated ERK. LCH lesions are associated with a local cytokine storm and exhibit a heterogeneous collection of inflammatory infiltrates (eosinophils, neutrophils and lymphocytes). The critical role of the immune system in regulating tumor growth and progression has been highlighted with the extraordinary impact of dis-inhibiting cytotoxic T cells in some adult cancers. However, the mechanisms behind tumor formation, both accumulation of pathologic CD207+ DCs as well as recruitment of inflammatory infiltrate in LCH, remain to be defined. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in approximately 75% of LCH cases, including recurrent BRAF -V600Eand MAP2K1 mutations. However, the inflammatory infiltrate within LCH lesions is composed of cells without any BRAF mutation or ERK activation. We hypothesized that the pathologic inflammation in LCH is orchestrated by interactions between the pathologic MAPK-activated DCs and normal immune cells. Defining the nature and function of infiltrating T cells in LCH has the potential to not only provide insights into mechanisms of pathogenesis and potential therapeutic targets, but may also inform novel physiologic mechanisms of DC-mediated T cell function. Clonality, investigated by exploring T cell receptor beta (TCRB) rearrangement in LCH patients, identified clonal populations among LCH lesion-infiltrating CD3+ T cells. The complementarity-determining region 3 (CDR3) of the TCRBV07 gene demonstrated marked monoclonal expansion, suggesting a potential involvement of antigen selection in the pathogenesis of LCH. High dimensional mass cytometric analysis identified multiple tumor-infiltrating T lymphocyte subsets in LCH. Functional analysis indicated hypercytokinemia and defective alloreactive CD8 effector T cells. Spanning tree progression of density normalized events (SPADE) analysis of infiltrating CD3+ T cells revealed high expression of T cell immunoglobulin mucin (TIM-3), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to healthy controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in LCH patients. In addition we found pathologic DCs in LCH lesions expressing ligands for the inhibitory receptors (PDL-1/L2, Galectin-9 and MHC Class II). We also found that patients harboring BRAF -V600E mutation express higher ligand expression compared to patients with wild type BRAF . Checkpoint inhibitor therapy by inhibiting PD-1 in an ex vivo setting to reinvigorate exhausted T cells only partially rescued CD8 effector cells alloreactivity in LCH patients. We are currently evaluating the efficacy of combination therapy in restoring effector T lymphocyte function. The results from this study thus support a model in which LCH lesion DCs with activated MAPK signaling select and recruit activated T cells with potential for clonal expansion to LCH lesions. While the CD207+ DCs in LCH lesions is histologically uniform across the disease spectrum, the composition of the immune microenvironment is variable. The variability of the inhibitory receptor ligand expression in BRAF -V600E vs BRAF wild-type lesions suggests that specific mechanisms of MAPK activation may have some distinct impact on T cell recruitment and activation. Finding more effective ways to reinvigorate T cells and have them regain functions could prove to be incredibly useful for efficacious therapeutic intervention in patients with LCH. DisclosuresNo relevant conflicts of interest to declare.

KRAS AND BRAF mutation rates and survival outcomes in colorectal cancer in an ethnically-diverse cohort

Background: KRAS-mutant colorectal carcinoma (CRC) is resistant to anti-EGFR antibodies. Several studies compare the KRAS mutation rates between Caucasians and African Americans, but few studies have compared other ethnicities. Aims: To compare the KRAS and BRAF mutation rates, as well as overall survival rates, in CRC between ethnic groups. Additionally, we report on correlations between clinicopathological variables relevant to CRC. Methods: Clinicopathological data from 284 ethnically-diverse patients with CRC were retrospectively analysed at Liverpool Hospital (2014–2016). KRAS and BRAF mutations were detected using real-time PCR kits (from Qiagen). Ethnicity was determined using both country-of-birth and surname. Survival outcomes were available for 191 patients. Overall survival was defined as date-of-diagnosis to death from any cause. Patients still alive were censored at 50-months post-diagnosis. Results and conclusions: KRAS mutation frequency differed significantly between Caucasians and Asians (36.1% vs 56.4%, p=0.018), and Asians and South-Americans (56.4% vs 16.7%, p=0.016), but not between other groups (Middle-Easterners 45.7%). BRAF-V600E mutation frequency differed significantly between Caucasians and Middle-Easterners (16% vs 0%, p=0.029), but not between other groups (Asian 8.6%, South-American 16.7%). The 50-month survival rate did not differ between ethnicities (Caucasians 44%, Middle-Easterners 52%, Asians 58%, South-Americans 50%). Of patients who died, the median survival time was 28 months. Overall survival did not significantly differ between patients with wild-type and mutant KRAS. Tumours with BRAF-V600E mutations were more likely to display microsatellite instability (MSI) than tumours with wild-type BRAF (45.5% vs 8.3%, p<0.001). Tumours displaying MSI occurred more frequently in the right colon (p=0.007). KRAS mutations were more frequent in circumferential tumours (p=0.04), and tumours without vascular invasion (p=0.004).

Loss of CDX-2 expression is an independent poor prognostic biomarker in patients with early-stage deficient mismatch repair colorectal cancer.

To investigate the clinicopathological factors, molecular features, and prognostic implications associated with loss of Caudal-related homeobox transcription factor 2 (CDX-2) expression in colorectal cancer (CRC) patients. Immunohistochemistry for CDX-2 expression was performed on formalin-fixed, paraffin-embedded primary CRC tissue samples from 449 patients. Correlation between CDX-2 expression and clinicopathological and molecular characteristics was evaluated. Univariate and multivariate survival analyses were performed to determine the prognostic value of loss of CDX-2 expression. Of 449 patients, 84% were stage I-III. CDX-2-negative expression was identified in 18 of 441 (4.1%) patients. Loss of CDX-2 expression was more commonly found in patients with right-sided tumors rather than left-sided tumors (odds ratio [OR] = 3.57; p=0.009), deficient mismatch repair (dMMR) compared to proficient MMR (pMMR) (OR = 3.7; p=0.012), and BRAF mutation compared to BRAF wild type (OR = 8.06; p=0.002). Univariate analysis revealed that stage I-III CRC patients with loss of CDX-2 expression had significantly worse overall survival (OS) and disease-free survival (DFS) than those with positive CDX-2 expression (5-year OS = 33.3% vs. 74.6%, respectively; p<0.001, and 5-year DFS: 42.9% vs. 69.5%, respectively; p=0.004). Loss of CDX-2 expression remained significantly associated with worse OS compared to positive CDX-2 expression in multivariate analysis (hazard ratio [HR] = 2.4; 95% confidence interval [CI], 1.12-5.11; p=0.023). Loss of CDX-2 expression was found to be associated with right-sided tumor, dMMR status, and BRAF mutation. Moreover, loss of CDX-2 expression is a poor prognostic factor for OS in stage I-III, even among patients with dMMR tumors.

Volume tumor impact on melanoma survival assessed using Breslow density

Volume tumor impact on melanoma survival assessed using Breslow density

LGG-05. GENERATION OF NOVEL MOUSE MODELS FOR BRAF V600E MUTANT GLIOMAGENESIS TO GAIN MECHANISTIC INSIGHTS INTO TUMOR FORMATION AND PROGRESSION

BackgroundThe BRAF V600E mutation occurs in ~ twenty percent of histologically diverse pediatric gliomas and is the second most common mutation in pediatric low-grade gliomas (LGG). BRAF V600E expression in LGG with balanced CDKN2A is associated with a higher rate for progression than for BRAF V600E wildtype tumors, and despite adjuvant therapy, consisting of resection, radiation and chemotherapy. Progression invariably occurs in BRAF V600E mutant CDKN2A deleted gliomas, marking a high-risk group. Here, we aim to overcome the lack BRAF V600E mutant glioma models that allow for studies of stem and progenitor cells and the immune system ability to understand progression. MethodsWe develop novel immunocompetent, stem and progenitor cell-based mouse models for BRAF mutant gliomas, including genetically engineered mouse models (GEMMs), orthotopic glioma models derived from gliomas in GEMMs as well as in vitro models of those tumors. BRAF mutant mouse brains and cells were analyzed by immunofluorescence staining, flow cytometry, mass cytometry and RNA sequencing. ResultsOngoing model development studies indicate that BRAF V600E mutant gliomas in murine brain exhibit very similar neuroanatomical preferences to human gliomas. The BRAF V600E mutation exacerbates the heterogenous cell cycling pattern of normal neural stem and progenitors and expands a symmetrically dividing progenitor population. Cellular plasticity rather than cellular lineage hierarchy drives the generation of a therapy resistant stem cell pool. Transcriptomic analyses of neuroglial stem cells with induced BRAF V600E expression provide insights into mechanisms for neoplastic transformation and progression. ConclusionAnalyses of two independent BRAF V600E mutant mouse models provide novel insights into the role for tumor intrinsic factors, such as plasticity and stemness, and the tumor microenvironment in progression.

Impact of BRAF mutations on prognosis and immunotherapy response in microsatellite instability/mismatch repair deficient metastatic colorectal cancer: A systematic review and meta-analysis.

3557 Background: Mismatch repair deficient/microsatellite instability high (dMMR/MSI-H) colorectal cancer (CRC) defines a molecular subtype with distinct clinicopathologic characteristics including an excellent response to immunotherapy. Although BRAF mutations are established as a negative prognostic marker in CRC, whether they retain their negative prognostic impact in or alter the response to immunotherapy in dMMR/MSI-H CRC remains unknown. Herein, we present a systematic review and meta-analysis of the impact of BRAF mutations on the overall survival (OS) and immune checkpoint inhibitor (ICI) response in dMMR/MSI-H CRC. Methods: Studies published from inception to 26 January 2021 were searched in PubMed, Embase, and major conference proceedings (AACR, ASCO, and ESMO). Eligible studies included the following: 1) observational studies reporting outcomes based on BRAF mutation status in dMMR/MSI-H CRC patients and 2) experimental studies of ICI reporting outcomes based on BRAF mutation status in dMMR/MSI-H CRC patients. A summary hazard ratio (HR) was calculated for OS in BRAF mutated ( BRAFmut) vs. BRAF wild type ( BRAFwt) patients (pts) with the random effects meta-analysis (REM). A summary odds ratio (OR) was calculated for objective response rate (ORR) in BRAFmut vs. BRAFwt pts treated with ICI with the REM. Results: Database search conducted according to PRISMA guidelines found 4221 studies in total. Initial screening identified 30 studies and after full-text review, 9 studies (N = 4158 pts) were included for the meta-analysis of prognosis (analysis A) and 3 studies (N = 178 pts) were included for the meta-analysis of ICI response (analysis B). The outcome measures are summarized in the table below. Analysis A showed that in stage I-IV dMMR/MSI-H CRC pts, BRAFmut was associated with worse OS than BRAFwt (HR 1.57, 1.23-1.99). The heterogeneity was low (I2 = 21%). Subgroup analysis showed no significant difference in the prognostic impact of BRAF mutation status between stage IV only and stage I-IV CRC pts. Analysis B showed no difference in ORR (OR 1.04, 0.48-2.25) between BRAFmut vs. BRAFwt dMMR/MSI-H pts who received ICI. The heterogeneity was low (I2 = 0%). Conclusions: BRAF mutations retain their negative prognostic impact in dMMR/MSI-H stage I-IV and stage IV CRC but are not associated with differential ICI response. Limitations include the following: analysis A was based on retrospective studies; also, the impact of BRAF status on the survival outcome of ICI could not be assessed due to limited number of studies.[Table: see text]

Treatment responses and disease dynamics in patients with untreated metastatic colorectal cancer receiving bevacizumab-based sequential versus combination chemotherapy: Analysis of a phase 3 trial (AIO KRK0110, XELAVIRI study).

3571 Background: Early response parameters such as early tumor shrinkage (ETS), depth of response (DpR), and time to DpR represent exploratory endpoints that may serve as early efficacy endpoints and potential predictors of long-term outcome. We analyzed the association of these endpoints with bevacizumab-based sequential (initial fluoropyrimidines) versus combination (initial fluoropyrimidines plus irinotecan) chemotherapy within a randomized phase III trial. Methods: DpR (change from baseline to smallest tumor diameter), ETS (≥20% reduction in tumor diameter at first reassessment), and time to DpR (study randomization to DpR-image) were analyzed in the XELAVIRI-trial. Moreover, progression-free survival (PFS) and overall survival (OS) were evaluated with ETS as stratification parameter (ETS vs. no ETS) according to treatment arm, molecular subgroup, and sex. Results: 370 patients were available for analysis of early treatment response parameters. A higher rate of ETS (60.9% vs. 43.5%; p = 0.001) and significantly greater DpR (-40.0% vs. -24.7%; p &lt; 0.001) were observed in the initial combination compared to the sequential therapy arm, respectively. The improvement of ETS and DpR was pronounced in the subpopulation of RAS/ BRAF wildtype patients. Male in contrast to female patients significantly benefitted from initial combination treatment in terms of median DpR (male: -40.0% vs. -22.2%; p &lt; 0.001; female: -34.0% vs. -24.4%; p = 0.13) and rate of ETS (male: 64.8% vs. 40.2%; p &lt; 0.001; female: 52.5% vs. 49.3%; p = 0.73). Achievement of ETS correlated with improved survival irrespective of treatment arm (PFS: p &lt; 0.001; OS: p = 0.012) and molecular subgroup (PFS: p &lt; 0.001; OS: p &lt; 0.001). Whereas the survival benefit in male patients achieving ETS was statistically significant (PFS: p &lt; 0.001, HR 0.532 (0.409-0.692); OS: p &lt; 0.001, HR 0.574 (0.437-0.756)), there were no significant differences in PFS (p = 0.107) and OS (p = 0.965) of female patients depending on ETS. Conclusions: In the XELAVIRI trial, initial irinotecan-based combination therapy with bevacizumab improves ETS and DpR in mCRC patients. Improvement in early response parameters appears pronounced in patients with RAS/ BRAF wildtype tumors suggesting a high sensitivity to irinotecan-based treatment. ETS was predictive of PFS and OS regardless of treatment arm. This finding was rather driven by male than female patients, potentially indicating that ETS might be less predictive of long-term outcome in an elderly, female population.

BRAF and NRAS mutation status and response to checkpoint inhibition in advanced melanoma.

9558 Background: The ability to analyze tumor mutation profiles has altered the oncology treatment landscape over the past decades. However, little is known about the effect of specific gene mutations on the response to immune checkpoint inhibitors (ICIs) in patients with advanced melanoma. Methods: All unresectable stage IIIc and IV patients with BRAF V600, NRAS mutations and BRAF and NRAS wild-type patients treated with anti-PD-1 or ipilimumab-nivolumab between 2012 and 2020 were included from the Dutch Melanoma Treatment Registry, a nationwide population-based registry. Outcomes were objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). A Cox model was used to analyze the association of possible prognostic factors with PFS and OS. Results: In total 1358 first-line patients treated with anti-PD-1 and 524 treated with ipilimumab-nivolumab were included. Median follow-up was 25.6 months for anti-PD-1 treated patients and 16.3 months for ipilimumab-nivolumab treated patients. The highest ORR, in first-line, to anti-PD-1 was in patients who were BRAF and NRAS wildtype (50.2%), compared to BRAF V600 (43.8%) and NRAS mutated patients (49.8%). ORR to ipilimumab-nivolumab was highest in NRAS mutated patients (44.9%), while ORR was 39.5% for BRAF mutated patients and 40.3% for wild-type patients. Median PFS in the anti-PD-1 treatment regimen was significantly higher (p = 0.049) for double wild-type patients (16.7 months) patients than for BRAF mutated patients (9.9 months) and NRAS mutated patients (11.3 months). PFS was not significantly different (p = 0.11) in the ipilimumab-nivolumab treatment cohort, with a median PFS of 6.5 months for the wild-type group, 10.8 months for the BRAF group, and 9.1 months for the NRAS group. In the anti-PD-1 treated patients, median OS was significantly higher (p &lt; 0.001) for BRAF mutated patients (32.8 months) compared to NRAS (21.0 months) and wild-type patients (23.0 months). For ipilimumab-nivolumab treated patients, median OS was also significantly higher (p &lt; 0.001) for BRAF mutated patients (36.5 months) than for NRAS mutated patients (11.8 months) and wild-type patients (16.1 months). After adjustment for potential confounders, the presence of a BRAF mutation remained associated with lower PFS in the anti-PD-1 treatment cohort and better OS in both treatment cohorts. Higher age, higher ECOG score, elevated LDH levels, liver metastases and brain metastases were associated with worse survival. Conclusions: PFS in first-line PD-1 was significantly higher for double wild-type patients than for BRAF mutant and NRAS mutant patients. PFS in ipilimumab-nivolumab treated patients did not significantly differ between BRAF mutant, NRAS mutant and double wild-type patients. OS was significantly higher for BRAF mutant patients in both treatment strata, which is probably the result of the subsequent BRAF/MEK-inhibition treatment option in this group.

Liquid biopsy-driven anti-EGFR rechallenge in patients with metastatic colorectal cancer.

3577 Background: The rechallenge with EGFR inhibitors represents an emerging strategy for anti-EGFR pre-treated patients with RAS wild type colorectal cancer (CRC). Unfortunately definitive selection criteria for anti-EGFR rechallenge in this setting are lacking. Very recently RAS wild type status on circulating tumor DNA (ct-DNA) at the time of rechallenge along with already known clinical criteria emerged as a potential watershed for this strategy. In the present study we explored liquid biopsy-driven anti-EGFR rechallenge strategy in the clinical practice for patients with metastatic colorectal cancer. Methods: Ct-DNA from RAS and BRAF wild type metastatic CRC patients previously treated with an anti-EGFR containing therapy was analyzed for RAS/BRAF mutations with the aim to evaluate the rechallenge strategy with anti-EGFR. The ct-DNA was analyzed for RAS-BRAF mutations using pyro-sequencing (PyroMark Q24 MDx Workstation) and nucleotide sequencing (Genetic Analyzer ABI3130) assays. Real-time PCR (Idylla) and droplet digital PCR (QX200 System) were performed to confirm the RAS-BRAF mutation status. Several clinical variables including previous response to anti EGFR containing therapy, tumor sidedness and anti-EGFR free interval were evaluated in relation to outcome. Tumor response evaluation was performed according to RECIST 1.1. Differences between categorical variables were evaluated using the Fisher’s exact test. Survival probability over time was estimated by the Kaplan–Meier method. Significant differences in the probability of survival between the strata were evaluated by log-rank test. Results: Twenty patients were included in the study. All patients were tested for RAS-BRAF mutations in ct-DNA. Fourteen patients (70%) showed a RAS-BRAF WT molecular profile, six patients (30%) showed a KRAS mutation. All the patients with ct-DNA RAS-BRAF WT profile underwent rechallenge with anti-EGFR. In details 11 patients (78.6%) underwent irinotecan+ cetuximab treatment, whereas 3 patients (21.4%) underwent panitumumab monotherapy. As for the outcome results to the rechallenge strategy, the median OS was 7 months (95% CI 5.0 to 13.0), the median PFS was 3 months (95% CI 2.0 to 6.0), the ORR was 27.3% with a DCR of 54.5%. Among the clinical variables evaluated as putative predictive/prognostic factors, previous response to anti-EGFR treatment was related to a not statistically significant improved OS (12 months vs 5 months HR:0.19 p: 0.06) and to a statistically significant improved ORR (75% vs 0% p:0.03). Conclusions: The rechallenge strategy with anti-EGFR confirmed to be feasible in clinical practice. The clinical outcome resulted consistent with the literature data. In addition to the molecular selection through the analysis of ct-DNA for RAS, previous response to anti EGFR treatment is confirmed as a prospective selection criteria for this therapeutic option.

A phase I study of SHR7390 plus camrelizumab in advanced/metastatic colorectal cancer.

e15553 Background: PD-1 antibody monotherapy has shown durable response in colorectal cancer (CRC) patients (pts) with microsatellite instability (MSI) high status, who account for only 10–15% of all CRC pts. Preclinical studies revealed that inhibition of MAPK pathway with MEK inhibitors can increase T-cell infiltration into tumors and therefore might enhance the antitumor activity of immune checkpoint inhibitor. Thus, this study was conducted to evaluate the safety and antitumor activity of SHR7390, a MEK1/2 inhibitor, plus camrelizumab (anti-PD-1) in pts with CRC. Methods: In this single-arm, open-label, phase I study, pts with treatment-refractory advanced or metastatic CRC were enrolled to receive SHR7390 in an accelerated titration scheme at doses of 0.125, 0.25, and 0.5 mg orally qd, plus camrelizumab at a fixed dose of 200 mg IV q2w. Eligible pts were given a single dose of SHR7390, observed for 7–10 days, and then treated continuously with SHR7390 and camrelizumab in 28-day treatment cycles until disease progression, intolerable toxicities, or withdrawal of consent. A recommended dose for expansion was determined based on the safety and tolerability of the dose escalation stage. The primary endpoints were dose limiting toxicity (DLT) and maximum tolerated dose (MTD). This study is registered with ClinicalTrials.gov, number NCT03182673. Results: At data cutoff on May 30, 2020, 22 pts were enrolled. The median follow-up duration was 6 months. Seven pts had received ≥3 lines of prior treatment. MSI status were available in 21 pts (MSS/MSI-L, n = 18; MSI-H, n = 3). There were two pts each in the SHR7390 0.125 and 0.25 mg cohorts, and 0.5 mg cohort was expanded to 18 pts. One DLT (grade 3 rash) was reported in a pt in the 0.5 mg cohort and the MTD was not reached. Grade 3–4 treatment-related adverse events (TRAEs) were rash (n = 4), increased lipase, oedema peripheral, face oedema, and anemia (n = 1 each). One pt reported a grade 5 TRAE (increased intracranial pressure). Five pts (23%) achieved partial response (0.125 and 0.25 mg cohorts, n = 1 each; 0.5 mg cohort, n =3), including one out of three pts with MSS/MSI-L &amp; BRAF mutant tumors (response lasting 13 months), one out of 15 pts with MSS/MSI-L &amp; BRAF wild type tumors (response lasting 8 months), and all three pts with MSI-H tumors (responses lasting 31, 11, and 10 months, respectively). Three pts with MSS/MSI-L achieved stable disease, and the overall disease control rate reached 36%. Conclusions: SHR7390 plus camrelizumab showed a tolerable safety profile. Preliminary clinical activity was reported regardless of BRAF and MSI status, and future studies are warranted to further evaluate these results. Clinical trial information: NCT03182673.

KRAS, NRAS and BRAF mutational status and first line treatment patterns in Slovenian population with metastatic colorectal carcinoma: Five year results.

e15546 Background: A phase IV non-interventional study was performed from 2013 till 2018 including 650 patients with primary aim to assess KRAS, NRAS and BRAF mutational status in Slovenian population with metastatic colorectal carcinoma (mCRC) suitable for first-line treatment. The evaluation of decisions for first-line treatment regarding the biomarkers status and assessing the possible impact of the time period of the biomarker status analysis report on the treatment decision were also incorporated in the analysis. The molecular analyses for KRAS and NRAS gene mutations were performed on exons 2, 3 and 4, and for BRAF gene mutations on exon 15. The first line systemic treatment options for RAS (KRAS/NRAS) wild type (wt) and mutated type (mt) mCRC subjects were as follows: chemotherapy - Fluoropyrimidine based systemic therapy combined with oxaliplatin and/or irinotecan with/without VEGF inhibitor bevacizumab and for RAS wt subjects, with/without EGFR inhibitors, cetuximab or panitumumab. Methods: To indicate the degree of certainty of KRAS, NRAS and BRAF status frequency as being wild type or mutant type 95% confidence interval was calculated. Results: The KRAS/NRAS/BRAF mutation rates were as follows - The distribution of subjects with KRAS mutated and wild-type tumors, was almost equal, 48.8% and 47.9% respectively. Eighty nine percent of the subjects had NRAS wild type tumours and 86.1% had BRAF wild type tumours. The most frequently used treatment regardless the biomarkers status and in accordance with the treatment guidelines was bevacizumab based combination therapy (53.1%). The EGFR inhibitor (cetuximab or panitumumab) based combination therapy was used in one third of mCRC subjects (30.9%), all with mCRC RAS wt. The time period from the initial presentation of the patient until the biomarker status analysis report was two weeks. Conclusions: With this study, we have proven that the distribution of the mutations in exons 2-4 of KRAS and NRAS genes and exon 15 in the BRAF gene in the Slovenian population with metastatic colorectal cancer matches historical data. Based on this, we conclude that the treatment decision in Slovenian population with metastatic colorectal carcinoma should be in the accordance with international treatment guidelines and on evidence based medicine. The molecular analysis performed at the Institute of Oncology Ljubljana was providing necessary biomarkers status report in an acceptable time that didn’t affect the treatment decision or delay the needed cancer treatment.

Prevalence of NTRK1/3 fusions in mismatch repair-deficient (dMMR)/microsatellite instable (MSI) tumors of patients with metastatic colorectal cancer (mCRC).

e15537 Background: Inhibitors of tropomyosin receptor kinase (TRK) have shown promising activity against neurotrophic TRK ( NTRK) fusion-driven cancers, regardless of tumor histotype or cell of origin. NTRK gene fusions are observed in less than 1% of colorectal cancers (CRCs). CRCs harboring wild-type BRAF and KRAS and MisMatch Repair deficiency (dMMR)/MicroSatellite Instability (MSI) due to MLH1 hypermethylation have been associated with NTRK fusions in small cohorts of non-metastatic tumors. We aimed at evaluating the incidence of NTRK fusions among dMMR/MSI metastatic CRCs (mCRC) for which there is a need for innovative therapies, as well as the associated clinical characteristics of these patients (pts) carrying NTRK fusion-positive tumors. Methods: Tumor samples of dMMR/MSI mCRC pts, paired primary and metastasis or primary alone, were obtained from a French multicenter retrospective cohort and from a single-center cohort of patients treated by immune checkpoint inhibitors (ICI) (Saint-Antoine Hospital, Paris). Clinico-pathological data including KRAS and BRAFV600E status, MMR proteins and MLH1 methylation status were available for all pts. All samples were screened for TRK expression by immunohistochemistry (IHC) using a pan-TRK antibody (clone EPR17341, Abcam; positivity: 1% of labeled tumor cells)) and for NTRK1 and NTRK3 gene rearrangements, by fluorescent in situ hybridation (FISH). A threshold of 15% nuclei positive for a break apart signal was considered positive for gene rearrangement. Results: A total of 158 pts with dMMR/MSI mCRCs (paired samples: n=39; primary only: n=119) were screened. Tumor samples of 10 patients (6.3%) harbored NTRK fusion genes by FISH ( NTRK1=8; NTRK3=2). Only four of these 10 patients had TRK immunoreactivity. One patient showed a discordance between metastasis harboring NTRK1 fusion (+) and primary tumor being negative. Eight tumors were sporadic with MLH1 hypermethylation. The remaining 2 cases were related to a MMR gene germline mutation (Lynch syndrome) with concurrent loss of MSH2 and MSH6 expression and isolated loss of MSH6 respectively. One Lynch-related tumor was KRAS mutated, one sporadic MLH1-negative tumor was BRAF V600E mutated. Four patients out of 91 treated by ICI had tumors with NTRK fusions. Three have shown radiological response according to iRECIST criteria (two complete responses, one partial response with 25 to 54 months of follow up) and one had primary resistance to ICI. Conclusions: Frequency of NTRK1/3 fusions is 6.3% in our dMMR/MSI mCRCs population. These fusions are not restricted to sporadic cases. The diagnostic accuracy of pan-TRK IHC is low. Optimal testing algorithms for theragnostics remain to be defined in this setting.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 &lt; 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.