BRAF Wt Patients Research Articles

Predictive Impact of Tumor Mutational Burden on Real-World Outcomes of First-Line Immune Checkpoint Inhibition in Metastatic Melanoma.

The choice of threshold and reliability of high tumor mutational burden (TMB) to predict outcomes and guide treatment choice for patients with metastatic melanoma receiving first-line immune checkpoint inhibitor (ICI) therapy in the real world is not well known. Using a deidentified nationwide (US-based) melanoma clinicogenomic database, we identified a real-world cohort of patients with metastatic melanoma (N = 497) who received first-line monotherapy anti-PD-1 (n = 240) or dual anti-PD-1 and anti-CTLA-4 ICI (n = 257) and had a tissue-based comprehensive genomic profiling test TMB score. TMB-high (TMB-H; ≥10 mutations per megabase [muts/Mb], n = 352, 71%) was independently predictive of superior real-world progression-free survival and overall survival versus TMB-low (<10 mut/Mb, n = 145, 29%) in both mono ICI (hazard ratio [HR], 0.45 [95% CI, 0.32 to 0.63]; P < .001; HR, 0.61 [95% CI, 0.41 to 0.90]; P = .01, respectively) and dual ICI (HR, 0.67 [95% CI, 0.49 to 0.90]; P = .009; HR, 0.61 [95% CI, 0.42 to 0.88]; P = .007, respectively) patients. Dual ICI offered no significant advantage in BRAFwt patients and unexpectedly demonstrated greatest benefit in the TMB 10-19 mut/Mb group, identifying a TMB-very high (≥20 mut/Mb, n = 247, 50%) BRAFmut patient subgroup for whom mono ICI may be preferable. TMB-H predicts superior outcomes on ICI while coassessment of BRAF status and TMB may inform first-line regimen choice.

Clinical Outcomes Following an Initial Stereotactic Radiosurgery Course for Brain Metastases from Melanoma

Brain metastases (BM) are common in melanoma patients. The effect of gene mutations is not well characterized since first-line metastatic therapy has shifted from chemotherapy (CHT) to molecularly targeted therapies (TT) and immunotherapy (IO). We report outcomes of melanoma BM patients stratified by molecular subtype and pre-stereotactic radiosurgery (SRS) systemic therapy. We identified all patients completing an initial SRS course for BM at two institutions between 1/2015 and 12/2020. Patients who had prior WBRT and/or resection were eligible. Demographic and clinical parameters were collected, along with melanoma tumor molecular characteristics. Intracranial progression (ICP) was defined as any radiographic distant and/or in-field progression per multidisciplinary consensus. Overall survival (OS) and freedom from ICP (FFICP) were estimated via the Kaplan Meier method. From a total of 1383 SRS BM patients, we identified 118 (8.5%) with melanoma. Median follow up was 8.7 months, median age 64 years (IQR 51-72), 81% had cutaneous origin, and 55% had a KPS of 90-100. Molecular subtypes included BRAF (45%), NRAS (9.3%), and c-KIT (3.4%). Overall, 61% received IO prior to SRS, while 25% and 9.3% received TT and CHT prior to SRS respectively. 60% of patients harboring a mutation received IO as first line therapy, 10% received TT, and 30% received both TT and IO prior to SRS. BRAFmut patients more likely to have received TT prior to SRS (43% vs 9.2%, p<0.001) compared to BRAFwt patients. Median OS was 9.7 months (95% CI 7.8-13) and was not significantly different from non-melanoma patients (p = 0.6). Median FFICP was worse for melanoma patients (5.9 mos, 95% CI 3.5-8.5) than non-melanoma patients (8.96 mos, 95% CI 8.2-9.7, p = 0.009). A total of 72 ICP events occurred, with 56 (77.8%) distant ICP cases, 3 (4.2%) in-field ICP, and 13 (18%) ICP events that were radionecrosis (RN) only. RN was associated with the presence of a targetable mutation (18% vs 2%, p = 0.006) and receipt of TT pre-SRS (36% vs 9.8%, p = 0.001). BRAFmut patients had significantly worse FFICP (3.8 mos, 95% CI 3.0-6.8) compared to BRAFwt patients (8.5 mos, 95% CI 5.8-30.2, p = 0.006), although median OS was not significantly different (9.6 mos, 95% CI 6.9-16 vs 10.7 mos, 95% CI 6.7-15.5, p = 0.8). NRASmut was associated with better FFICP (29 mos, 95% CI 2.94-NA, p = 0.02). In this modern, multi-institutional cohort of SRS patients, melanoma BM patients had worse FFICP compared to non-melanoma BM patients, and BRAFmut patients had worse FFICP than BRAFwt patients. RN was associated with mutational status and receipt of TT pre-SRS. OS did not vary significantly across groups. This analysis may help inform systemic therapy decisions and future genomic studies for patients with BMs from melanoma.

Maintenance strategies after anti–EGFR-based induction in RAS wild-type (wt) metastatic colorectal cancer (mCRC): A systematic review and Bayesian network meta-analysis.

148 Background: In RAS wt mCRC the use of maintenance therapy after induction with fluoropyrimidine (FP)-based cytotoxic therapy (CT) plus anti-EGFR agents is controversial. Herein, we conducted a systematic review and network meta-analysis to compare different maintenance strategies. Methods: PubMed, CENTRAL, Embase and oncological meeting websites were screened to identify eligible studies. Phase II-III randomized trials which enrolled patients with previously untreated RAS wt mCRC, who switched to a maintenance strategy after an induction of CT combined with an anti-EGFR agent were included. Maintenance strategies considered were: observation, single agent anti-EGFR or FP, FP + anti-EGFR, doublet CT + anti-EGFR. Outcomes were progression-free survival (PFS), overall survival (OS) and safety (grade 3-4 adverse events). Subgroup analyses were performed in BRAF wt patients and left sided tumors. The meta-analysis was conducted using a random-effects model. A Markov Chain Monte Carlo simulation was used to estimate the posterior distributions. Strategies were ranked using the surface under the cumulative ranking (SUCRA) probabilities. Risk of bias was assessed with Cochrane Risk of Bias Tool. Results: Overall, 4207 records were screened and 10 trials were included in the analysis. Cumulative risk of bias was low-moderate. Maintenance therapy with FP + anti-EGFR (HR 0.56, 95% CrI 0.36-0.88) and doublet CT + anti-EGFR (HR 0.68, 95% CrI 0.46-1.01), showed the greatest PFS benefit compared to observation. On SUCRA analysis, FU + anti-EGFR had the highest likelihood of improving PFS (96.3%), compared to doublet CT + anti-EGFR (73.5%) or EGFR monotherapy (48.0%). In terms of OS, compared to observation, maintenance therapy with doublet CT + anti-EGFR (HR 0.51, 95% CrI 0.19-1.28), FU + anti-EGFR (HR 0.59, 95% CrI 0.19-1.68) or anti-EGFR monotherapy (HR 0.56, 95% CrI 0.24-1.34) yielded similar results. On SUCRA analysis probabilities were 78.3% with doublet CT + anti-EGFR, 64.5% with FP + anti-EGFR and 63.4% with anti-EGFR monotherapy. FP + anti-EGFR confirmed to be the best option in terms of PFS in BRAF wt patients (SUCRA: 96.3%) and left sided tumors (SUCRA: 89.6%). OS results in BRAF wt patients were consistent with the primary analysis. In left sided tumors, the OS benefit of adding CT seems limited compared to EGFR monotherapy. The addition of CT to anti-EGFR agents resulted in an increase of toxicity while FP monotherapy plus anti-EGFR showed a favourable safety profile compared to doublet CT + anti-EGFR. Conclusions: FP + anti-EGFR should be considered the best maintenance option in RAS wt mCRC in terms of PFS. Taking into account the similar OS performance of the different strategies, EGFR monotherapy could be considered, especially in left-sided tumors. Doublet CT + anti-EGFR, despite being effective, is limited by toxicities.

Real-world comparison of outcomes in the first line treatment of metastatic melanoma according to BRAF mutation status: Nivolumab and ipilimumab therapy vs. nivolumab monotherapy.

e21519 Background: In metastatic melanoma (MM) patients, nivolumab can be used as monotherapy or in combinations with ipilimumab. The combination therapy has been shown in a randomized clinical trial to provide survival benefit in BRAF mutant patients with an increased toxicity. Using real-world data, we evaluated the impact on overall survival and post-treatment hospitalizations of 1L nivo alone vs nivo+ipi. Methods: We performed a retrospective cohort study of 1L treatment of patients with MM between 1/2016 and 12/2020, utilizing administrative claims data from the Anthem Cancer Care Quality Program. Real-world overall survival (OS) was defined as time from diagnosis to death. A Cox model with inverse probability of treatment weighting (IPTW) was used to adjust for demographic and clinical features. Adjusted hazard ratios (aHR) were estimated using weighted Cox proportional hazards models. Results: Our cohort included 708 1L MM patients, of them 466 (66%) treated with nivo+ipi. There was no difference in BRAF status between the treatment groups. Patients treated with nivo+ipi were younger and had more evidence of brain and lung metastasis. Following adjustment, nivo treated had significant longer OS compared to the nivo+ipi treated (aHR: 1.73 (1.25-2.35), p = 8e-4). This difference mostly stems from increased mortality of the combo treated in the first 6 months. At 24-months, 58% of the combination therapy treated patients were alive compared to 74% in the monotherapy group (p = 0.0006). Survival benefit was only observed in BRAF WT patients. In V600E patients no survival difference was observed (aHR: 0.8 (0.4-1.6), p = 0.5). Patients treated with the combination therapy had 5-fold more hospitalizations during the first 90 days after the treatment start (46% vs 10%, p = 3e-22). Conclusions: Our retrospective cohort study demonstrated improved survival for the monotherapy in BRAF WT patients. Combination therapy was associated with significantly more treatment-related hospitalizations and deaths, mainly in the first 6 months. The increased early morbidity of the combo should be considered in treatment decision.[Table: see text]

Treatment patterns and outcomes following disease progression on anti-PD-1 therapies for advanced melanoma.

Background: Anti-PD-1-based therapies prolong survival in advanced melanoma, but disease progression is common. This study evaluated treatment patterns and overall survival (OS) after anti-PD-1 progression. Methods: Retrospective data from patients with advanced melanoma and progression on anti-PD-1 treatment between 2014 and2019 were taken from Flatiron Health, which reflects largely community practice. Treatment patterns and OS were analyzed for BRAF mutant (mt) and wild-type (wt) subgroups; OS was also examined across all patients. Results: Progression following anti-PD-1 was recorded for 679 patients. Median OS ranged from 5.0 to11.3months. Of 275 BRAFmt and 374 BRAFwt patients, 113 (41.1%) and 228 (61.0%) received no subsequent therapy, respectively. However, 48.4% of BRAFmt and 57.8% of BRAFwt patients continued anti-PD-1 treatment beyond progression. Conclusion: This real-world study underscores the need for effective treatments for advanced melanoma post-progression on anti-PD-1 therapy.

Dynamics in treatment response and disease progression of metastatic colorectal cancer (mCRC) patients with focus on BRAF status and primary tumor location: analysis of untreated RAS-wild-type mCRC patients receiving FOLFOXIRI either with or without panitumumab in the VOLFI trial (AIO KRK0109)

PurposeIn mCRC, disease dynamics may play a critical role in the understanding of long-term outcome. We evaluated depth of response (DpR), time to DpR, and post-DpR survival as relevant endpoints.MethodsWe analyzed DpR by central review of computer tomography images (change from baseline to smallest tumor diameter), early tumor shrinkage (≥ 20% reduction in tumor diameter at first reassessment), time to DpR (study randomization to DpR-image), post-DpR progression-free survival (pPFS = DpR-image to tumor progression or death), and post-DpR overall survival (pOS = DpR-image to death) with special focus on BRAF status in 66 patients and primary tumor site in 86 patients treated within the VOLFI-trial, respectively.ResultsBRAF wild-type (BRAF-WT) compared to BRAF mutant (BRAF-MT) patients had greater DpR (− 57.6% vs. − 40.8%, p = 0.013) with a comparable time to DpR [4.0 (95% CI 3.1–4.4) vs. 3.9 (95% CI 2.5–5.5) months; p = 0.8852]. pPFS was 6.5 (95% CI 4.9–8.0) versus 2.6 (95% CI 1.2–4.0) months in favor of BRAF-WT patients (HR 0.24 (95% CI 0.11–0.53); p < 0.001). This transferred into a significant difference in pOS [33.6 (95% CI 26.0–41.3) vs. 5.4 (95% CI 5.0–5.9) months; HR 0.27 (95% CI 0.13–0.55); p < 0.001]. Similar observations were made for patients stratified for primary tumor site.ConclusionsBRAF-MT patients derive a less profound treatment response compared to BRAF-WT patients. The difference in outcome according to BRAF status is evident after achievement of DpR with BRAF-MT patients hardly deriving any further disease control beyond DpR. Our observations hint towards an aggressive tumor evolution in BRAF-MT tumors, which may already be molecularly detectable at the time of DpR.

BRAF V600E-mutated anaplastic thyroid carcinoma (ATC) and treatment with BRAF-inhibitors: Real-world data from a single-institution, still far from the cure.

e18577 Background: ATC is a very aggressive disease. In the last years new insights on molecular profile of ATC have led to novel therapeutic options, primarily BRAF-targeted agents. Methods: From May 2018 to December 2019, data on recurrent/metastatic ATC patients treated at our Institution have been reviewed. BRAF V600E was assessed in each patient. BRAF mutation was tested both on tumor sections and on cell-free DNA (cfDNA). Circulating BRAF V600E was assessed by using Droplet Digital PCR (ddPCR) Technology. BRAF wild-type (WT) patients were treated with standard chemotherapy (adriamicin or taxans or platinum-based regimens). BRAF V600E patients were treated with dabrafenib 300 mg per day and trametinib 2 mg per day (combo regimen) until progression or unacceptable toxicities. Response was assessed by RECIST 1.1; progression-free survival (PFS) and overall survival (OS) were estimated with Kaplan-Meier method and compared with log-rank test. Safety was evaluated according to CTCAE version 5.0. Results: 16 ATC patients were included. Male/female 5/11, median age was 68 years (range 43-80). Eight patients (50%) carried BRAF mutation. Best response in BRAF V600E was 4 partial response (PR), 3 stable disease (SD) and response in 1 patient has not been assessed yet. Combo was well tolerated, the most common adverse events (G2) were fatigue (14%), pyrexia (14%), nausea and vomiting (9%). Only one patient experienced drugs interruptions. Best response in BRAF WT patients was SD for 1, progressive disease for 4 and 3 patients received supportive care. Concordance between circulating BRAF status with tumor tissue was 80%. Assessment of circulating BRAF performed at baseline and at best response was available for 3 patients achieving a PR: mean reduction was -99.48% from baseline (range -99.37 – -99.66%). Median follow up was 22.8 weeks (95% CI 12.8-22.8). Median PFS was 13 weeks in BRAF V600E and 7.4 weeks in BRAF wt patients (p=0.1); in the same groups median OS was 18.4 and 7.8 weeks, respectively (p=0.051). Conclusions: Real-life data with combo regimen showed an ORR of 50%, with short PFS and OS. Reduction of circulating BRAF observed during treatment might be an indirect marker of tumor shrinkage, even if more data are needed to correlate circulating BRAF and disease response.

Real-world treatment patterns and clinical outcomes of advanced melanoma patients following disease progression on anti-PD-1-based therapy.

e22036 Background: Despite the success of anti-programmed cell death-1 (PD-1) based therapies that prolong survival in advanced melanoma, some patients experience disease progression. Real-world treatment patterns and outcomes after progression on anti-PD-1 based regimens are unknown. Methods: Adults with advanced melanoma and a record of disease progression on anti-PD-1 treatment (alone or in combination) between 1 Sept 2014–31 Jan 2019 were selected from the Flatiron Health Oncology electronic medical record (EMR) database for this retrospective study. Index progression date was defined as an event where the treating clinician concluded in the patient record that there had been disease progression. The first subsequent therapy received was identified. Kaplan-Meier methods estimated overall survival (OS) with 95% confidence interval (CI) after the progression date. Analyses were run separately for BRAF mutant (mt) and wild type (wt). Results: Among 2,169 patients with advanced melanoma treated with an anti-PD-1, 213 BRAFmt and 302 BRAFwt had a record of progression following anti-PD-1 initiation and were included; an additional 82 BRAFmt and 138 BRAFwt were excluded due to death without a record of progression. Median age was 64 and 71 years, respectively; 27.7% and 9.9% had &gt;1 line of therapy prior to the anti-PD-1. Among the BRAFmt patients who progressed on anti-PD-1, 94 (44.1%) received no subsequent therapy, 80 (37.6%) BRAF±MEK (± IO), and 16 (7.5%) anti-CTLA4 alone or with anti-PD-1 after progression on the initial anti-PD-1; 11% received other treatments with no clear pattern of care. Median (95% CI) OS from the index progression date for BRAFmt was 11.9 (8.2–16.9) months. In a subgroup of these BRAFmt patients with BRAF treatment prior to or during the first anti-PD-1 line (N = 65), 52.3% had no subsequent therapy and a median OS of 4.0 (2.7–7.5) months. Among the BRAFwt patients who progressed on anti-PD-1, 186 (61.6%) received no subsequent therapy, 46 (15.2%) received anti-CTLA4 alone or with anti-PD-1, 26 (8.6%) switched anti-PD-1s, and 15% received other treatments. Median (95% CI) OS for BRAFwt was 10.1 (8.8–12.5) months. Conclusions: In this real-world retrospective study, &gt; 40% of BRAFmt and &gt; 60% of BRAFwt patients did not initiate a new regimen after progression on anti-PD-1 therapy. Median OS after progression was &lt; 1 year. This may be overestimated, as it excluded patients who died before progression was recorded in the EMR. These findings highlight a need for more effective treatments for advanced melanoma.

Proteomic test for anti-PD-1 checkpoint blockade treatment of metastatic melanoma with and without BRAF mutations

The therapeutic landscape in metastatic melanoma has changed dramatically in the last decade, with the success of immune checkpoint inhibitors resulting in durable responses for a large number of patients. For patients with BRAF mutations, combinations of BRAF and MEK inhibitors demonstrated response rates and benefit comparable to those from immune checkpoint inhibitors, providing the rationale for sequential treatment with targeted and immunotherapies and raising the question of optimal treatment sequencing.Biomarkers for the selection of anti-PD-1 therapy in BRAF wild type (BRAF WT) and in BRAF mutated (BRAF MUT) patients help development of alternative treatments for patients unlikely to benefit, and might lead to better understanding of the interaction of checkpoint inhibition and targeted therapy. In this paper we evaluate the performance of a previously developed serum proteomic test, BDX008, in metastatic melanoma patients treated with anti-PD-1 agents and investigate the role of BRAF mutation status. BDX008, a pre-treatment proteomic test associated with acute phase reactants, wound healing and complement activation, stratifies patients into two groups, BDX008+ and BDX008-, with better and worse outcomes on immunotherapy.Serum samples were available from 71 patients treated with anti-PD1 inhibitors; 25 patients had BRAF mutations, 39 were wild type. Overall, BDX008+ patients had significantly better overall survival (OS) (HR = 0.50, P = 0.016) and a trend for better progression-free survival (PFS) (HR = 0.61, P = 0.060) than BDX008- patients. BDX008 classification was statistically significant in the analyses adjusted for mutation status, LDH, and line of treatment (P = 0.009 for OS and 0.031 for PFS). BRAF WT BDX008+ patients had markedly long median OS of 32.5 months and 53% landmark 2 years survival, with statistically significantly superior OS as compared to BDX008- patients (HR = 0.41, P = 0.032). The difference between BDX008+ and BDX008- in PFS in BRAF WT patients and in OS and PFS in BRAF MUT patients did not reach statistical significance, though numerically was consistent with overall results. The test demonstrated significant interaction with neutrophil-to-lymphocyte ratio (NLR) (PFS P = 0.041, OS P = 0.004). BDX008 as a biomarker selecting for benefit from immune checkpoint blockade, especially in patients with wild type BRAF and in subgroups with low NLR, warrants further evaluation.

Oncogenic alterations detected by droplet digital PCR in patients with metastatic colorectal cancer resistant to cetuximab.

575 Background: Anti-EGFR therapy is the standard of care for metastatic colorectal cancer (mCRC) patients with RAS and BRAF genes wild type (wt). Secondary alterations of several genes have been identified as possibly resistant mechanisms to EGFR blockade, these including mutations of KRAS, NRAS, BRAF, MEK and EGFR ectodomain, as well as amplifications of HER2 and c-MET. In this study, we investigated alterations of these targeted genes for mCRC patients with acquired resistance to cetuximab treatment by using non-invasive droplet digital PCR (ddPCR) method within circulating DNA. Methods: We enrolled 38 RAS and BRAF wt patients, who progressed after failure of cetuximab contained regimens between Jul 2015 and Jan 2018. Plasma samples from all 38 patients were collected and detected for seven candidates (mutation of KRAS, NRAS, EGFR, BRAF, MEK, amplification of HER2, c-MET) by using ddPCR at the time of baseline, each evaluation by interval of 2 months and documented progression. All clinical parameters were collected simultaneously. Results: A total of 23 secondary alterations were found in 17 (17/38,44.7%) cetuximab resistant patients. The targeted gene alterations were detected as follows: 9 (9/23,39%) RAS mutations, 5 (22%) HER2 amplifications, 5 (22%) EGFR mutations, 2 (9%) c-MET amplifications, 1 (4%) BRAF and 1 MEK mutation. Among 17 patients, 6 patients had multiple alterations, including 2 patients with KRAS+EGFR mutations, 2 patients with HER2+c-MET co-amplifications and 2 patients with KRAS gene exon 2 and 3 multiple mutations. Primary sites were 15 left sides and 2 right sides, descending colon (12 cases) was the most common origins. Eleven patients were synchronous disease. Twelve patients received cetuximab as first line therapy, whereas 5 patients in the ≥ 2nd line setting. All of these 17 patients, plasma levels of oncogenic alterations detected by ddPCR were showed dynamic changing and good agreement with tumor responding status. Conclusions: Oncogenic alterations detected by ddPCR were found in almost half mCRC patients resistant to cetuximab. Dynamic changes of specific alteration may facilitate making decisions for selection of anti-EGFR mAb during the treatment course.

Streamlining universal screening for lynch syndrome (LS): Towards improved yield of genetic counseling (GC).

503 Background: LS is largely underdiagnosed although Universal Screening (US) in colorectal cancer (CRC) patients through MisMatch Repair deficiency (MMR-d) testing is widely endorsed. Low adherence to guidelines among oncologists may be partly due to a lack of consensus on whether all MMR-d patients should be referred to GC/Genetic Testing (GT). As BRAF mutation rules out LS, we estimated the increased yield of LS diagnosis from GC /GT which could be obtained by selecting candidates for GC through BRAF testing. Methods: From 2011 to 2016, 1447 consecutive stage I-IV CRC surgical patients at a single institution, underwent immunohistochemistry (IHC) for LS using anti MLH1, MSH2, MSH6 and PMS2 antibodies. Oncologists were invited to refer all MMR-d patients to GC/GT. BRAFV600E testing was carried out only in case of MLH1 protein loss at IHC. Results: MMR-d was found in 194 patients (13%), with 171 showing loss of MLH1 expression (88%). Oncologists referred 27 (16%) to GC. Among the 21 who underwent GC, BRAF testing and GT, 9 were BRAF wild type (wt) (43%) and none had LS. Among the 23 MMR-d patients with loss of expression of MSH2, MSH6 or PMS2 (≠MLH1), oncologists referred 9 to GC (39%): 7 underwent GC / GT and 3 carried LS (43%) at GT. Median age was 76 years (range 30-97) in the MMR-d group, 78 (range 41-97) in the MLH1 group and 63 (range 30-86) in the ≠MLH1 group. Overall, LS was diagnosed in 3 of the 28 MMR-d patients (11%) who underwent GC /GT, possibly an underestimate due to the advanced median age of our MLH1 loss patients. Had we only offered GC to the 9 BRAF wt patients among the 21 with MLH1 loss, we could have avoided 12 (57%) of the GC sessions conducted, increasing the yield of LS diagnosis from 3/28 (11%) to 3/16 (19%) (75% increase). Conclusions: When US for LS is adopted, a GC referral rate reduction of 57% among MLH1 loss patients, and an overall increase in the yield of GC of about 75% can be obtained by testing for BRAF mutation before oncologist referral to GC rather than after. As multistep selection of patients by oncologists may be unfeasible, CRC pathology reports with combined MMR-d and BRAF testing (for MLH1 loss at IHC) and an ‘LS suspicion alert’ could improve oncologists’ awareness of LS and compliance with guidelines.

Expression and correlation of phosphorylation extracellular signal-regulated kinase and monopolar spindle 1 in colorectal cancer with wild type or BRAF V600E mutation

Objective To determine the correlation between phosphorylation extracellular signal-regulated kinase (p-Erk) and monopolar spindle 1 (Mps1) in colorectal cancer patients with the BRAF V600E mutation or not, and to explore the relationship between the expression of p-Erk and Mps1 with the clinicopathological features and prognosis of colorectal cancer patients. Methods Two hundred and eighty-eight paraffin-embedded tissue sections containing both the carcinoma and its adjacent non-neoplastic colorectal tissue were collected from January 2009 to June 2015 in the First Hospital of Shanxi Medical University. BRAF mutation was detected by Sanger sequencing. Kaplan-Meier survival analysis was used to analyze the correlation between BRAF V600E and prognosis. Immunohistochemistry was used to detect the expression level of p-Erk and Mps1 in colorectal cancer with wild type or BRAF V600E mutation. The correlation between p-ERK and Mps1 expression was analyzed by using linear regression analysis. Results The BRAF V600E mutation rate was 5.2% in colorectal carcinomas. In addition, the poorly differentiated tumours and mucinous tumours had higher incidence of BRAF mutations than well differentiated tumours and non-mucinous tumours respectively [14.3% (9/63) vs. 2.7% (6/225), χ2 = 11.208, P = 0.001; 25.0% (6/24) vs. 3.4% (9/264), χ2 = 16.630, P <0.001). The positive rate of p-Erk and Mps1 in colorectal carcinomas with BRAF V600E was significantly higher than that in colorectal carcinomas with BRAF WT (14/15 vs. 3/15, P < 0.05; 15/15 vs. 3/15, P < 0.05]. It was found that the p-Erk expression correlated positively to the Mps1 expression (R 2 = 0.419, P < 0.001). The expressions of p-Erk protein in poorly differentiated adenocarcinoma was significantly higher and mucinous adenocarcinoma than those in high differentiated adenocarcinoma and non-mucinous adenocarcinoma (χ 2 = 6.679, P = 0.01; χ 2 = 5.735, P = 0.017), as well as in the group with lymph node metastasis than without lymph node metastasis (χ 2 = 5.436, P = 0.02). Positive rate of Mps1 in poorly differentiated carcinoma was higher than that in well differentiated adenocarcinoma of colorectal carcinomas (χ 2 = 7.950, P = 0.009). The Kaplan-Meier survival analysis indicated that patients with BRAF V600E had a worse survival rate than BRAF WT patients. Conclusions BRAF V600E may play an important role in specific pathological kinds of colorectal carcinomas, which is expected to be an independent prognostic factor. The expression of p-Erk is significantly correlated with Mps1 in colorectal carcinomas, suggesting that Mps1 may become a new potential target for targeted therapy. Key words: Colorectal neoplasms; BRAF V600E mutation; Phosphorylation extracellular signal-regulated kinase; Monopolar spindle 1

Dynamic monitoring HER2 amplification of circulating DNA in metastatic colorectal cancer patients treated with cetuximab.

714 Background: Addition of cetuximab has shown clinical benefit in RAS and BRAF wild-type (wt) patients with metastatic colorectal cancer (mCRC). Human epidermal growth factor receptor 2 ( HER2) amplification was reported to be one of the resistance mechanisms to cetuximab. We investigated whether monitoring of HER2 amplification in circulating DNA allows early detection of progression and informs about resistance to cetuximab. Methods: We analyzed HER2 amplification in circulating DNA by 8 week-interval using droplet digital polymerase chain reaction (ddPCR) from 30 RAS and BRAF wt patients, who progressed after failure of cetuximab-containing regimens between Jul 2015 and Jun 2017. Results: 16.7% (5/30) of patients exhibited dynamic fluctuations of HER2 amplification in plasma, one of whom was found to be positive for HER2 amplification in matched tumor tissue sample using FISH. Two patients had HER2 and c-MET co-amplification. All 5 primary sites were left side, 4 rectums and 1 descending colon. 3 patients received cetuximab as first line therapy, whereas 2 patients in the 2nd line setting. Among these 5 patients, changes in circulating DNA levels showed good agreement with changes in tumor volume. Furthermore, quantifications of HER2 amplification showed obvious increase with an average lead time of 2 months compared with CT documented progress. But interestingly, there was no difference in progression-free survival (PFS) between these 5 patients and the others without HER2 amplification (p &gt; 0.05). Conclusions: Plasma HER2 amplification detected by ddPCR was showed dynamic changing over time and would predict resistance to cetuximab-containing treatment. Average 2 months lead time was observed and need to validate in the further study.

Molecular alterations to predict survival and response to chemotherapy of pediatric low-grade glioma.

10503 Background: RAS/MAPK pathway mutations have been identified as the major drivers of pediatric low-grade glioma (pLGG). The impact of these alterations on outcome and response to therapy is still unknown. Methods: We performed a large population based study of all pLGG diagnosed from 1985-2015. Detailed treatment and very long term outcome data was collected on all patients. Known pLGG-related alterations were detected using NanoString and QX200™Droplet Digital™PCR. Molecular data was correlated with outcome and response to chemotherapy. Results: In our cohort of 614 patients, BRAF was found to be altered in 57% and wild-type (WT) in 43% of patients without neurofibromatosis 1 (NF1). Among BRAF-WT we identified H3.3K27M, FGFR1-TACC1, MYBL1 and other alterations. Molecular alterations stratified pLGG into several risk groups. Ten-year progression free survival (PFS) was 72.3% for NF1, 69.5% for KIAA1549-BRAF, 53.5% for BRAF-WT, 30.3% for BRAF-V600E and 0% for H3.3K27M mutations (p &lt; 0.0001). Similarly, overall survival (OS) at 10 years delineated difference between excellent survival of KIAA1549-BRAF and NF1 compared to BRAF-V600E and BRAF-WT (p = 0.0005). Interestingly, all patients with FGFR1-TACC1 and MYBL1 were alive despite observed progressions. Strikingly, response to chemotherapy determined by changes in tumor size at 6 months of therapy correlated with pLGG alteration. Objective response to first line chemotherapy was observed in 46% of patients with KIAA1549-BRAF and 35% of NF1. In contrast, only 15% BRAF-V600E and 18% BRAF-WT responded and 41% tumors grew after six months of chemotherapy. Moreover, 5-year PFS after chemotherapy was strikingly low for BRAF-V600E and BRAF-WT (25% and 31.9% respectively) compared to KIAA1549-BRAF (50%) and NF1 (76.7%) (p = 0.001). This translated to decreased OS for BRAFV600E and BRAF-WT patients (p = 0.042). Conclusions: Our study provides evidence that molecular alterations dictate the outcome of pLGG. KIAA1549-BRAF harbors excellent prognosis and choice of therapy should be made in favor of less toxic agents to minimize deleterious late effects. In contrast, poor prognosis is associated with lack of response to chemotherapy in BRAF-V600E and BRAF-WT tumors.

BRAF-V600 mutational status affects recurrence patterns of melanoma brain metastasis.

Brain metastasis is common and carries a poor prognosis in melanoma. A single institution, retrospective cohort of 225 melanoma patients was analyzed to determine if BRAF-V600 mutational status was associated with brain metastasis. Eighty-three of the 225 patients (37%) had BRAF-V600 mutations. At initial diagnosis, BRAF-V600 mutations were associated with younger age (p ≤ 0.001), higher proportion of females (p = 0.0037), higher AJCC stage (p = 0.030), regional lymph node involvement (p = 0.047), and family history of cancer (p = 0.044). Compared to BRAF-WT, BRAF-V600 patients had an increased risk of brain metastasis in multivariate analysis (OR = 2.24; 95% CL = 1.10-4.58; p = 0.027). However, BRAF-V600 patients treated with a selective BRAF inhibitor (BRAFi) had a similar risk of brain metastasis compared to BRAF-WT patients (OR = 1.00; 95% CL = 0.37-2.65; p = 0.98). Moreover, treatment with BRAFi significantly prolonged the time from initial diagnosis to brain metastasis diagnosis (HR = 0.30; 95% CL = 0.11-0.79; p = 0.015). Compared to other tissues, the brain was the most frequent site of metastasis in BRAF-V600 patients without BRAFi (42% ± 7%). The frequency of brain metastasis was lower in BRAF-WT and BRAF-V600 patients with BRAFi (25% ± 4% and 25% ± 8%, respectively). The proportion of patients with brain metastasis as the only site was 40%, 60%, and 0% in the BRAF-WT, BRAF-V600 without BRAFi, and BRAF-V600 with BRAFi groups, respectively. This study provides evidence on the clinical importance of BRAF-V600 mutations and BRAF inhibition in the progression to melanoma brain metastasis.

EGFR methylation and outcome of patients with advanced colorectal cancer treated with cetuximab

Targeted therapy of metastatic colorectal cancer (mCRC) with monoclonal antibody anti-epidermal growth factor receptor (EGFR) agents, such as cetuximab (CTX) or panitumumab, is the treatment strategy of choice in patients characterised by a wild-type (wt) RAS gene status. However, despite selection based on RAS status, a high proportion of patients do not respond to therapy. EGFR methylation has been reported to have a role in predicting the response to anti-EGFR agents. The present study aimed to evaluate the role of EGFR methylation in association with the clinical outcome of patients with mCRC treated with CTX. In total, 64 patients with mCRC were assessed in the present study. Genomic DNA was extracted from tumoral tissue and EGFR methylation and mutation of the KRAS, BRAF and PIK3CA genes were analysed by pyrosequencing. EGFR expression was assessed by immunohistochemistry. The various alterations were analysed by assessing the objective response rate (ORR), progression free survival (PFS) and overall survival (OS) rates. In total, 42 cases (66%) exhibited >10% EGFR methylation and there was no correlation with EGFR expression. Mean EGFR methylation of 41 and 9% was observed in KRAS-mutated and -wt patients, respectively (P=0.05). Conversely, a high EGFR methylation was observed in BRAF-wt patients with compared with patients possessing the mutated gene (18 vs. 3%, respectively; P=0.07). EGFR methylation was significantly correlated with the OS rate [hazard ratio, 0.98; 95% confidence interval (CI), 0.96–1.00; P=0.019], but not PFS rate. In patients with a methylation rate <10 and >10%, the median OS rate was 7.5 months (95% CI, 4.4–9.4 months) and 12.0 months (95% CI, 8.7–13.9 months), respectively (P=0.034). In conclusion, the present study revealed a correlation between EGFR methylation and improved OS rate in patients treated with CTX-based chemotherapy. The presence of EGFR methylation is inversely correlated with BRAF and PIK3CA mutations, indicating that the prognostic value of gene methylation may be worth verifying in further studies.

Pharmacodynamic effects of regorafenib in metastatic colorectal carcinoma (mCRC).

e14507 Background: Regorafenib, an oral multikinase inhibitor of VEGFR2/3, PDGFRb, KIT, FGFR, RET, RAF and TIE2, is efficacious in refractory mCRC but its mechanism of action is unclear and predictive biomarkers are lacking. Methods: We assessed tumor and circulatory biomarkers in a phase 2 study of regorafenib in refractory mCRC patients. Regorafenib was administered orally at 160mg/d for 3 out of 4 weeks. Post cycle 2 response was assessed by RECIST 1.1. Subjects were scheduled for FDG PET-CT scans (pre + D15) and paired core needle tumor biopsies for IHC analysis (pre + D21) in cycle 1. Archival tumor mutations were evaluated using Sequenom MassARRAY OncoCarta Panel V1.0 assay. Results: 35 patients were treated; 49% received &gt; 4 prior therapies and 43% had prior bevacizumab. Median PFS was 3.45 mths (95% CI: 3.40-3.49), ORR was 3% and disease control rate [DCR] (PR + SD at 8 wks) was 57%. Early PET responses (EORTC criteria) were seen in 49%, but did not predict for DCR (p=1.0). Fatigue, hand foot skin reaction (HFSR), voice change and diarrhea occurred in &gt; 30% of subjects. Grade 3-5 toxicities occurred in 46%, the commonest being HFSR and rash (17% each). Median relative dose intensity was 92%; 43% required &gt; 1 dose reduction, 60% required &gt; 1 dose interruption. KRAS (29%), BRAF (9%), EGFR (9%), NRAS (6%) KIT (3%), PIK3CA (3%), PDGFRA (3%) and CDK (3%) mutations were detected in archival tumors. None predicted for ORR or DCR; PFS was identical in KRAS mutant vs wt patients (3.45 mths, p=0.39) and similar in BRAF mutant vs wt patients (3.48 vs 3.45 mths, p=0.10). The patient with the longest PFS (12.6 mths) had a BRAF mutation. Amongst the 10 paired tumor samples available, IHC markers upregulated in &gt;50% cases were pMEK, pERK, pJun and pJNK, whilst those downregulated/ unchanged in &gt;50% were pKIT, pVEGFR2, CD31 [vascular endothelial cells (ECs)] and podoplanin (lymphatic ECs). The greatest change was observed in podoplanin expression, corresponding to a 60% reduction in lymphatic vessel density. Conclusions: FDG-PET responses in cycle 1 did not predict for regorafenib clinical benefit in mCRC patients. Targeting lymphatic/vascular ECs in the tumor microenvironment may be a more significant antitumor mechanism of regorafenib than MAP kinase pathway inhibition. Clinical trial information: NCT01189903.

P1.28 Dusp4 Expression as A Marker of Heterogeneous Signaling in Colorectal Cancer Patients

ABSTRACT Introduction Previous analysis of the gene expression data of mutational subgroups in the PETACC3 trial, revealed a very homogenous gene expression pattern in BRAF mutant CRC patients; while KRAS mutant CRC contain at least three different patient groups in terms of gene expression. One of these KRAS mutant groups together with 10% of KRAS/BRAF WT (WT2) patients, resembled the gene expression found in the BRAF mutants. This BRAF-like subgroup is characterized by poor prognosis. In this article we investigate, if heterogeneity in CRC patients can be partly explained by diverse signaling downstream of KRAS, to which the DUSPs take part. Material and methods The expression of DUSP1, DUSP4 and DUSP6, DUSPs previously shown to be induced by mutations in genes of the MAPK pathway, was analyzed in 4 different CRC patient microarray datasets (PETACC3, cetuximab, TCGA and Agendia dataset). We investigated, if patients with low and high DUSP expression behave differently in terms of response, OS, PFS and differ in clinical characteristics both in PETACC3 and TCGA dataset. Findings Unlike DUSP1, DUSP4 and DUSP6 were differentially overexpressed between BRAF mutants and KRAS mutants on one side and between BRAF mutants and WT2 patients on the other side. When we subdivided the patients into low and high DUSP expression within KRAS mutant and WT2 patients, we found a significant difference in OS in both mutational subgroups with high DUSP4 expression in KRAS mutants associated with a better OS and in WT2 associated with a worse OS. Multivariate analysis in the BRAF WT patients in both PETACC3 and cetuximab dataset showed that high DUSP4 expression was still associated with a worse OS (HR = 1.23; p Conclusions DUSP4 is predictive for OS in CRC patients with KRAS mutations. Interestingly, within KRAS mutant and WT2 patients high DUSP4 expression correlates with clinical characteristics that are typical for BRAF mutant CRC patients. If DUSP4 may represent a novel biomarker for a subset of CRC patients needs further research. Download : Download full-size image Fig. 1 . Box plots with DUSP4 expression according to different mutational groups in PETACC3 dataset. Download : Download full-size image Fig. 2 . Kaplan-Meier curve for overall survival according to DUSP4 expression (low and high) in KRAS WT patients.

Analysis of BRAF mutation status in high-risk malignant melanoma patients having received HD IFNa therapy in adjuvant setting.

e19020 Background: Mutation in BRAF gene is considered to be a predictor of response to vemurafenib. However, it still remains to be demonstrated wheter BRAF mutational status is a prognostic factor in high risk melanomas. Methods: Between May 2003 till July 2011, there were 100 patients with a wide local excision of high risk malignant melanoma removed, who received HD IFNa adjuvant treatment. We retrospectively analyzed the data of 57 patients, who had determined BRAF V600 mutation status by real-time PCR assay (Cobas 4800 BRAF V600 Mutation Test). 48 patients were identified as a high-risk melanoma initialy, 9 after locoregional recurrence. Results: We have analysed samples from primary tumors (N35), lymph nodes (N16) and mets (N6). BRAF V600 mutation was found in 35 cases (61.4%). The median age was 52 yrs (range 19-66 yrs) in the whole group, in the subgroup of BRAF MT/WT it was 52.5/51 yrs (nonsignificant). The female/male ratio was 22/35, with no sex dependent differences for the BRAF MT or WT. The most frequent tumor stage T4N0M0 (29.8%) was the most common indication to the IFN treatment. The recurrence (distant mets) of melanoma was observed in 33 patients (57.8%); BRAF MT/WT 21 vs 12, HR 0.74, (95%CI 0.37-1.48) p =0.25. We observed 24 deaths (42.1%); BRAF MT/WT 16 vs 8, HR 0.72 (95%CI 0.32-1.62) p =0.46. We found a statistically significant higher risk of death associated with lower age in the whole group (p =0.006; OR =0,92). Logistic regression in the subgroup of BRAF MT patients revealed that the positivity of lymph nodes was associated with 5.6-times higher probability of death (OR =0.89, p =0.094). Additionally, a higher age was significantly associated with better overall survival (OS) p=0.012. On the other hand, in the subgroup of BRAF WT patients neither the age nor the positivity of lymph nodes influenced OS. Conclusions: We confirmed that BRAF mutation occurs in approximately 60% high-risk malignant melanomas. There were no statistically significant differences in DFS or OS between BRAF MT/WT form. However, our analysis corroborates the notion that BRAF MT patients have higher risk of death or recurrence, which we consider to be clinically relevant.

New insights to gene expression signatures from primary FFPE tumors for the prediction of response to cetuximab in KRAS and BRAF wild-type colorectal cancer (CRC).

3588 Background: Cetuximab - an anti-EGFR monoclonal antibody improves overall and progression-free survival (PFS) in a subset of advanced-CRC patients. It has been demonstrated that KRAS and BRAF mutations are negative predictors of response to cetuximab and it has become standard to reserve treatment for KRAS wild-type (wt) tumors. However, there is an important proportion of KRAS and BRAF wt patients that do not respond to cetuximab. Our study aimed at identifying markers for response prediction to cetuximab in KRAS and BRAF wt patients via gene expression profiling. Methods: A CRC specific DNA microarray platform (CRCDSA) designed to work with formalin-fixed paraffin-embedded (FFPE) tissue was used for expression profiling of 65 FFPE CRC primary tumor samples. 44 KRAS and BRAF wt patients were selected for the analysis. We fitted a Cox proportional hazards model on PFS (42 events) in order to identify genes related to increased risk of progression after cetuximab treatment. Results: In the top 100 genes significantly associated with progression, the FGFR2 receptor was identified suggesting the activation of a signaling pathway alternative to EGFR. Other interesting genes connected to increased risk of progression were CTSE, S100B, MUC2, APC. Genes negatively correlated with progression were TOE1, XIAP, NAIP (inhibitors of apoptosis). Some of the genes (VAV3, EREG, PLAGL2, DIDO1,YES1) were in agreement with results of the same analysis we have performed on a publicly available dataset derived from metastatic tissues (Khambata-Ford S et al, JCO 2007). Conclusions: By using a transcriptome-focused approach to analyze FFPE primary tumor samples, we have identified novel targets and pathways of potential relevance to response to cetuximab in BRAF and KRAS wt tumors. This could be of significance in the development of new therapeutics for KRAS and BRAF wt nonresponsive patients and demonstrates the usefulness of FFPE primary material for response prediction of metastatic disease. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Almac Diagnostics Merck Serono