BRAF are potentially targetable alterations in a wide variety of solid cancers Zoran Gatalica1*, Ken Burnett1, Ryan Bender1, Rebecca Feldman1, Semir Vranic2, Sandeep Reddy1 1 Caris Life Sciences, Phoenix, AZ, USA, 2Department of Pathology, Clinical Center, University of Sarajevo, Sarajevo, Bosnia and Herzegovina *Corresponding author: Zoran Gatalica, [email protected]

Abstract (updated) Methods Results, contd. Results, contd. Background: The BRAF mutations are potentially targetable with several Tissue samples Figure 3. Potential therapeutically relevant alterations that co-occur with BRAF treatment modalities approved in a limited number of cancer types. The present Table 1. Frequency of V600 Mutations across Tumor Types V600_ PIK3CA pathway RAS pathway A cohort of 36,312 solid tumors were profiled at Caris Life Sciences, (Phoenix, AZ) using Sanger Histoytpe V600E V600K V600R V600E(2) V600D V600M V600_ K601delinsE study explored the spectrum and frequencies of BRAF mutations in a wide variety W604delinsR BRAF Status EGFR pos. PTEN loss AKT PIK3CA PTEN STK11 KRAS HRAS NRAS V600E 58% (148/255) 49% (536/1046) 2% (8/532) 8% (50/653) 6% (31/509) 2% (9/521) 0.2% (2/927) 0.4% (2/465) 1% (4/705) and NGS assays. Next-generation sequencing (NGS) and Sanger sequencing: CRC 578 - - - - - 1 - of solid tumors. other V600 17% (5/30) 44% (55/98) 1% (1/76) 1% (1/90) 6% (4/68) 1% (1/71) 0% (0/89) 0% (0/65) 0% (0/90) NGS was performed on enriched tumor genomic DNA isolated from formalin-fixed paraffin 462 82 12 3 1 1 3 - non-V600 42% (84/201) 35% (246/378) 1% (5/337) 8% (28/367) 7% (21/322) 5% (15/323) 19% (73/393) 2% (6/303) 5% (20/369) Materials and Methods: 36,312 solid tumors were profiled using different gene embedded samples using the Illumina MiSeq platform. Specific regions of the genome were TC 75 ------V600E vs. other V600 (K,R,D) 0.0001 ns ns 0.0142 ns ns ns ns ns V600E vs. non-V600 0.0007 sequencing assays (Sanger and Next-generation sequencing, Caris Life Sciences, amplified using the Illumina TruSeq Amplicon - Cancer Panel. The NGS panel included 45 NSCLC 58 2 - - - - 2 1 0.0001 ns ns ns 0.018 0.0001 ns 0.0001 CUP 20 1 ------75% Phoenix, AZ) and immunohistochemistry. different listed here: http://www.carismolecularintelligence.com/next-generation- EOC 40 ------Results: Overall, 5% of all solid tumors harbored BRAF mutations (77% V600E and sequencing-profile). All variants were detected with >99% confidence based on the frequency of GBM 24 ------50% % Altered 23% non-V600E). As expected, BRAF mutations were detected in 38% of thyroid the present and the amplicon coverage using a mutation frequency threshold of 10% NET 9 ------25% Biliary Tract 9 ------cancers (4% non-V600E), 35% malignant (13% non-V600E), 9% (4, 5). All regions that were sequenced achieved a minimum of 100x coverage and overall Sarcomas 7 ------0% V600E other V600 non-V600 samples had an average coverage of >500x. Sanger Sequencing was used to explore selected Breast 6 - - - - 1 - - colorectal (10% non-V600E), 6% small intestinal cancers (92% non-V600E) and 4% EGFR positive PTEN loss AKT PIK3CA PTEN STK11 KRAS HRAS NRAS NSCLC (65% non-V600E). Non-V600E mutations with functional impact on regions of BRAF, KRAS, c-KIT, EGFR, and PIK3CA. Testing also included one or more of the ECD* 4 ------following: expression (IHC), gene amplification (C/FISH), microsatellite instability testing Uterine 3 ------Figure 3. Co-occurring alterations in BRAF-mutated patients, according to specific BRAF alterations. EGFR activity included (% variant/non-V600E): V600K (24%), D594G (7%), G469A (9%), Gastric 3 ------positivity and PTEN loss are detected by IHC, whereas AKT, PIK3CA, PTEN, STK11, KRAS, HRAS and NRAS by fragment analysis (FA). Pancreatic 3 - 1 - - - 1 - G466V (6%), and G469V (5%). Some other cancer types also harbored BRAF are detected by sequencing methods. Positive EGFR status was significantly higher in V600E solid tumors Low Grade Glioma 2 ------than other V600 mutations (V600K/R/D, etc.) and non-V600 mutations, as well. PIK3CA was found at mutations (GBM; 75% V600E and 25% non-V600E, biliary tract cancers; 43% GIST 1 ------higher frequency in V600E and non-V600, than in V600K/R/D, etc. KRAS occurred at significantly higher **Full analysis involved an additional 11293 patients profiled since abstract submission SBA 1 ------V600E, 57% non-V600E), while esophageal/GEJ, gastric, liver, pancreatic and head frequency in non-V600 BRAF-mutated tumors than V600E. Results *Erdheim-Chester Disease 4/4 V600E-mutated and neck cancers were mostly devoid of BRAF mutations. The most common 50

45 concurrent alterations associated with potential resistance to BRAF inhibitors, and 90% Tumor Type (BRAF-mutated/n, n=total tested tumor type) Prevalence of BRAF mutations, % Figure 4. Comparison of co-occurring with implications for dual-targeting approaches, included mutations of PIK3CA 80% V600E non-V600 40 alterations in BRAF-mutated colorectal (7%), PTEN (6%), KRAS (5%), NRAS (2%), AKT1 and NRAS (1%, respectively), and 0 5 10 15 20 25 30 35 40 70% 35 cancers. RAS mutations are more frequent **Thyroid Carcinoma (78/206) 37.9% 60% over-expression of EGFR (53%). Of interest, overexpression of EGFR in BRAF- 50% in non-V600, however 4 BRAF V600E Melanoma (670/1929) 30 mutated tumors was highest in the tumor type with poor clinical response to BRAF 34.7% 40% mutated CRC tumors harbored concurrent Colorectal Adenocaricnoma (641/7222) 8.9% 25 30% activating RAS mutations: (KRAS: Q61L, inhibitors (colorectal cancer with 80%), and lowest in melanoma (6%) where 20%

Frequency of of Alterations Frequency G13D; NRAS:V45I, HRAS:c.290+2T>C). MSI Small Intestinal Adenocarcinomas (13/216) 5.6% monotherapy has been more successful. 20 10% was more frequent in V600E mutated CRC Non-Small Cell (175/4037) V600-Mutated Non-V600 Mutated 4.3% 0% compared to non-V600-mutated CRC (36% Conclusions: Activating BRAF mutations (both V600E and non-V600E) are 15 EGFR KRAS HRAS NRAS Microsatellite PIK3CA Cancers of Unknown Primary (CUP) (43/1079) 3.9% Instability vs. 6%). 10 potentially targetable in a substantial proportion of various solid malignancies. A Glioblastoma (32/930) 3.4% subset of BRAF-mutated cancers harbors additional genetic alterations and/or Biliary Tract Cancers (21/703) 3.0% 5 over-express EGFR which may require expanded/dual targeting treatment to Low Grade Glioma (2/88) 2.3% 0 L4P

BRAF P14L

mutations were not detected in the following E26D L584I T589I I592T I463T K601I L584F F583L S446L L597S S467L F595L G469I N581I F468S L597P L597R L597V E586K K601E V471F R443T R462T T599R H585L R603X T599A S605G G469S L597Q D594E N581S G460E G464E G469E G466E N581T F468S A305V G466K G469K G596C E695Q K601N H585R G596R G466R G464R G469R D594V V528D K601Q Q461K D594A G596V G464V G466V G469V G460A G466A G469A R603Q R444Q D594H D594G D587N D594N (blank) D449fs G469S G466E G466V G469A R444W D594N G606W overcome resistance. Neuroendocrine Tumors (16/823) 1.9% T599dup Conclusions

tumor types: hepatocellular carcinomas (0/277), G30_A33del T599_V600del V487_T491del A33_A34insAP A598_T599insI Kidney Cancer (6/341) 1.7% A598_T599insT Activating BRAF mutations (both V600E and non-V600E) are potentially A598_V600delinsRP •

thymic carcinomas (0/54) and uveal melanomas W450_G460delinsPL

c.1517+2_1517+3ins9 Small Cell Lung Cancer (4/261) 1.5% (n=0/104). Figure 2. Frequency of non-V600 mutations across solid tumors. targetable in a substantial proportion of various solid malignancies.

Gastric Adenocarcinoma (6/401) 1.5% Background **Non-V600 mutations in thyroid cancer (TC) were Epithelial (81/5883) 1.4% The BRAF gene, located on 7q34, is a constitutive part of the K601E (n=2) in follicular variant of papillary TC, and Table 2. Most frequent non-V600 mutations are described with preclinical and clinical implications • The V600E mutations were more prevalent (77%) although a substantial Most-observed Clinical (C) or Pre-clinical (P) Data Soft Tissue Tumors (Sarcomas) (10/976) 1.0% F583L in papillary TC Variant Citations proportion of non-V600E mutations with functional impact on kinase activity MAPK/ERK signaling pathway involved in cancer initiation and progression. BRAF is tumor type Regarding Treatment Implications Bladder Cancer (4/385) 1.0% G469A NSCLC G469A: Porcelli et al. (2015) Journal of Translational Medicine, was also detected: V600K (24%), D594G (7%), G469A (9%) G466V (6%) and one of the most frequently mutated genes in human cancer (~7%) (1). Since 2002 Gastrointestinal Stromal Tumors (GIST) (1/113) 0.9% G469V NSCLC C/P : Likely resistant to 13( Suppl 1):P3 C: Potentially responsive to sorafenib G469L: Gautschi, et al. (2013) Lung Cancer, 82(2):365-7. G469V (5%). when Davies et al. described BRAF mutations in a subset of human (2) 0.8% G469R Melanoma, CRC Prostatic Adenocarcinomas (3/383) G469R: Sereno, M, et al. (2015) Anticancer Drugs, 26(9):1004-7. there have been numerous studies exploring BRAF status in human neoplasms Pancreatic Adenocarcinoma (10/1331) 0.8% D594N NSCLC P: kinase dead mutation, utility to BRAF inhibitors are not supported, however, if found in the presence of • A subset of BRAF-mutated cancers harbors additional genetic alterations (e.g. melanoma, thyroid, colorectal, lung, ovarian cancer, hairy cell leukemia, Uterine/Endometrial Cancers (28/3224) Heidorn, et al. (2010) Cell 140(2):209-21. 0.8% D594G CRC activated RAS, then MEK inhibitors may be of potential benefit. (KRAS, PIK3CA, PTEN, AKT1, NRAS) and/or over-express EGFR, which may multiple myeloma, histiocytoses) (3-4). Breast Cancer (19/4082) 0.6% C: Likely responsive to vemurafenib L597R: Bahadoran , et al. (2013) J Clin Oncol, 31(19) CRC, Melanoma, require expanded/dual targeting treatment to overcome a potential resistance L597R/S C: Likely responsive to MEK inhibition (TAK-733, L597S: Dahlman, et al. (2012) Cancer Discovery, 2:791-797. Gastroesophageal (GE)/GEJ cancer (2/543) 0.5% Ovarian ) L597Q : Bowyer, et al. (2014). Melanoma Res, 24(5):504-8. mechanism. The BRAF gene (V600E and non-V600E) mutations are clinically relevant due to the Non-Epithelial Ovarian Cancer (1/263) 0.4% P: Slightly higher levels of BRAF signaling compared G464V CRC, NSCLC with wildtype BRAF, however vemurafenib lacks Yang, et al. (2010) Cancer Res 70, 5518. Head & Neck Cancer (1/458) 0.2% targeted treatment modalities (e.g. vemurafenib, dabrafenib, tramatenib) that activity against this alteration. Melanoma, P: low kinase activity compared to V600E, however have been approved in a limited number of cancers so far (3-4). G466E References Ovarian still activates ERK through CRAF, sorafenib is a better Wan, et al. (2004) Cell, 116(6):855-867. Figure 1. BRAF mutation spectrum across solid tumors. Of 36,312 solid tumors G466V NSCLC inhibitor of CRAF than mutant BRAF 1. Jabbar KJ et al. Am J Surg Pathol 2015;39:454-61. N581I NSCLC P: low kinase activity compared to V600E, however 2. Davies H et al. Nature 2002;417:949-54. In the present study we explored the spectrum and frequencies of BRAF mutations tested, 1867 harbor BRAF mutation, for a mutation frequency of 5%. Frequency still activates ERK through CRAF, sorafenib is a better Wan, et al. (2004) Cell, 116(6):855-867. N581S Uterine, Ovarian 3. Hall RD, Kudchadkar RR. Cancer Control 2014;21:221-30. in a large cohort of solid tumors and analyzed other potentially targetable BRAF inhibitor of CRAF than mutant BRAF displayed is % of mutations among total tested, across tumor types. Blue and K601E Melanoma, NSCLC C: Likely responsive to MEK inhibition (trametinib) K601E: Bowyer, et al. (2014). Melanoma Res, 24(5):504-8. 4. Hyman, DH et al. NEJM 2015;373:726-736. biomarkers co-existing with BRAF mutations. Red bars indicate the distribution of V600- and non-V600 mutations across tumor 5. Gatalica Z et al. Oncotarget 2015;6:19819-25. types, respectively.