Analysis of KRAS/NRAS and BRAF mutations in the phase III PRIME study of panitumumab (pmab) plus FOLFOX versus FOLFOX as first-line treatment (tx) for metastatic colorectal cancer (mCRC).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3511-3511 ◽  
Author(s):  
Kelly S. Oliner ◽  
Jean-Yves Douillard ◽  
Salvatore Siena ◽  
Josep Tabernero ◽  
Ronald L. Burkes ◽  
...  
Keyword(s):  
Braf Mutation ◽  
Predictive Biomarker ◽  
Primary Objective ◽  
Phase Iii ◽  
Braf Mutations ◽  
Exon 2 ◽  
Mutation Status ◽  
Exon 4 ◽  
Kras Exon ◽  
Clinical Trial Information

3511 Background: Analysis of a phase III pmab monotherapy study indicated that KRAS and NRAS mutations beyond KRAS exon 2 may be predictive of pmab efficacy (Peeters et al, 2013). Methods: The primary objective of this prospectively defined retrospective analysis of PRIME was to assess the effect of pmab + FOLFOX vs FOLFOX on overall survival (OS) in pts with mCRC based on RAS (KRAS or NRAS) or BRAF mutation status. "Gold standard" bidirectional Sanger sequencing and WAVE-based SURVEYOR Scan Kits from Transgenomic (conducted independently) were used to detect mutations in KRAS exon 3, exon 4; NRAS exon 2, exon 3, exon 4; and BRAF exon 15. Results: RAS ascertainment rate was 90%. Tx HRs for pts with WT RAS were 0.78 (95% CI, 0.62 - 0.99; p = 0.04) for OS (median gain of 5.8 months in the pmab arm) and 0.72 (95% CI, 0.58 - 0.90; p = < 0.01) for PFS. Tx HRs for WT KRAS exon 2/mutant (MT) other RAS were 1.29 (95% CI, 0.79 - 2.10; p = 0.31) for OS and 1.28 (95% CI, 0.79 - 2.07; p = 0.32) for PFS. Tx HRs for pts with WT or MT BRAF were inconsistent with a predictive biomarker (Table). Prognostic effects of the tested biomarkers will be presented. Conclusions: A statistically significant OS benefit was observed in pts with WT RAS mCRC treated with pmab + FOLFOX vs FOLFOX. Pmab is unlikely to benefit pts with any RAS mutations. In this analysis, BRAF mutation had no predictive value. Clinical trial information: NCT00364013. [Table: see text]

Analysis of KRAS/NRAS mutations in PEAK: A randomized phase II study of FOLFOX6 plus panitumumab (pmab) or bevacizumab (bev) as first-line treatment (tx) for wild-type (WT) KRAS (exon 2) metastatic colorectal cancer (mCRC).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3631-3631 ◽  
Author(s):  
Lee Steven Schwartzberg ◽  
Fernando Rivera ◽  
Meinolf Karthaus ◽  
Gianpiero Fasola ◽  
Jean-Luc Canon ◽  
...  
Keyword(s):  
Retrospective Analysis ◽  
Progression Free Survival ◽  
Primary Analysis ◽  
Exon 2 ◽  
Free Survival ◽  
Randomized Phase Ii ◽  
Exon 4 ◽  
Grade 3 ◽  
Kras Exon ◽  
Clinical Trial Information

3631 Background: PEAK estimated the tx effect of FOLFOX6 with pmab or bev in 1st-line WT KRAS mCRC. The PRIME study showed significantly improved progression free survival (PFS) and overall survival (OS) with pmab + FOLFOX vs FOLFOX in pts with WT RAS (KRAS/NRAS exons 2, 3, 4) mCRC in a prospective-retrospective analysis (unpublished data). Methods: This prospective-retrospective analysis of PEAK was designed to assess the effect of pmab + FOLFOX6 or bev + FOLFOX6 on PFS (primary endpoint) and OS in WT RAS (KRAS/NRAS exons 2, 3, 4) mCRC. Pts were required to have WT KRAS exon 2 tumors. Bidirectional Sanger sequencing and Transgenomic SURVEYOR/WAVE analysis were independently conducted to detect mutations in KRAS exon 3 (codons 59/61), exon 4 (codons 117/146); NRAS exon 2 (codons 12/13), exon 3 (codons 59/61), exon 4 (codons 117/146); BRAF exon 15 (codon 600) in banked specimens. Results: 285 WT KRAS (exon 2) mCRC patients (pts) were randomized, 278 received tx. The current RAS ascertainment rate is 75%. Tx HRs (pmab:bev) for pts with WT RAS were 0.63 (95% CI, 0.43-0.94; p = 0.02) for PFS and 0.55 (95% CI, 0.30-1.01; p = 0.06) for OS (Table). The incidence of worst grade 3-5 adverse events was consistent with the primary analysis. Updated OS and BRAF results will be presented. Conclusions: In this 1st-line estimation study in WT RAS mCRC, PFS and OS HR favored pmab + FOLFOX6 relative to bev + FOLFOX6, suggesting that activating RAS mutations appear to be predictive for pmab tx effect. The safety profile for both arms was consistent with previously reported studies. Clinical trial information: NCT00819780. [Table: see text]

Analysis of KRAS/NRAS mutations in phase 3 study 20050181 of panitumumab (pmab) plus FOLFIRI versus FOLFIRI for second-line treatment (tx) of metastatic colorectal cancer (mCRC).

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. LBA387-LBA387 ◽  
Author(s):  
Marc Peeters ◽  
Kelly S. Oliner ◽  
Timothy Jay Price ◽  
Andres Cervantes ◽  
Alberto F. Sobrero ◽  
...  
Keyword(s):  
Progression Free Survival ◽  
Primary Objective ◽  
Primary Analysis ◽  
Exon 2 ◽  
Ras Mutation ◽  
Mutation Status ◽  
Free Survival ◽  
Ras Genes ◽  
Ras Status ◽  
Kras Exon

LBA387 Background: Previously, this study showed significant improvement in progression-free survival (PFS) in pmab + FOLFIRI vs FOLFIRI (HR=0.73; 95% CI: 0.59-0.90; p=0.004) and a trend toward improved overall survival (OS; HR=0.85; 95% CI: 0.70-1.04; P=0.12; Peeters et al. JCO 2010). Recently, analysis from 1st-line mCRC PRIME study showed that mutations in RAS genes (KRAS/NRAS exons 2/3/4) predicted a lack of response to pmab (Douillard et al. NEJM 2013). Methods: The primary objective was to assess the tx effect of pmab + FOLFIRI vs FOLFIRI on OS and PFS based on RAS mutation status in the primary analysis population. Bidirectional Sanger sequencing was used to detect mutations in KRAS exons 3, 4 and NRAS exons 2, 3, 4 in patients (pts) with known WT KRAS exon 2 mCRC. Results: In this prospective retrospective analysis, overall RAS ascertainment rate was 85% (n=1008/1186). 18% of the WT KRAS exon 2 pts harbored additional RAS mutations (n=107/597). Efficacy is shown (Table). Tx HR for pts with WT RAS was 0.803 (95% CI: 0.629-1.024; P=0.077) for OS and 0.695 (95% CI: 0.536-0.903; P=0.006) for PFS. Conclusions: Improvements were observed in the tx effect of pmab + FOLFIRI vs FOLFIRI on OS and PFS in the WT RAS group vs the WT KRAS exon 2 group. Pts with MT RAS mCRC are unlikely to benefit by the addition of pmab to FOLFIRI, similar to pts with MT KRAS exon 2 mCRC in this study. These findings are consistent with previously reported outcomes by RAS status and support RAS testing to determine potentially appropriate pts for pmab tx. Clinical trial information: NCT00339183. [Table: see text]

Expanded RAS and BRAF V600 testing as predictive biomarkers for single agent cetuximab in the randomized phase III CO.17 trial.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 537-537 ◽  
Author(s):  
Jonathan M. Loree ◽  
Anthony Dowers ◽  
Dongsheng Tu ◽  
Christopher J. O'Callaghan ◽  
Dan Edelstein ◽  
...  
Keyword(s):  
Single Agent ◽  
Predictive Biomarkers ◽  
Digital Pcr ◽  
Phase Iii ◽  
Molecular Profile ◽  
Braf Mutations ◽  
Exon 2 ◽  
Archival Tissue ◽  
Frequency Detection ◽  
Kras Exon

537 Background: KRAS/NRAS ( RAS) testing of exons 2, 3 and 4 is standard prior to anti-EGFR treatment in metastatic colorectal cancer and many consider BRAFV600 ( BRAF) mutations predictive. CO.17 was a randomized phase III trial comparing cetuximab vs best supportive care (BSC) in unselected patients (pts). Re-analysis tested only KRAS exon 2, thus the benefit of cetuximab in RAS/BRAF wild type (WT) pts is unclear. Methods: We retrospectively performed expanded RAS/BRAF testing using a highly sensitive digital PCR method (BEAMing; 1% allele frequency detection limit) on micro-dissected archival tissue from 248 CO.17 pts. Additional pts without available archival tissue, with prior Sanger sequencing or therascreen results were included in analyses if mutations were previously detected (n = 77). Overall survival (OS), progression free survival (PFS), and response rates (RR) were compared by molecular profile. Results: Of 248 sequenced pts, 139 (56%) were RAS mutant, with 112 (45%) exon 2, 11 (4%) exon 3 and 6 (2%) exon 4 KRAS mutant, and 10 (4%) NRAS mutant pts. Seven (3%) BRAF mutant, and 97 (30%) confirmed RAS/BRAF WT pts were identified. Results are summarized below. A test of interaction indicated RAS status was predictive for PFS (p = 0.0001) and OS (p = 0.037) and BRAF status neared significance as a predictive marker for PFS (p = 0.089) but not OS (p = 0.24). Conclusions: These updated results demonstrate an improved PFS (HR 0.25 vs 0.40 previously) and OS (HR 0.51 vs 0.55 previously) for cetuximab in RAS/BRAF WT pts compared to prior analyses that included only KRAS exon 2 mutation status. We provide an estimate of single agent cetuximab efficacy for future anti-EGFR re-challenge studies and demonstrate further support that BRAF mutations may predict lack of benefit from anti-EGFR therapy. Clinical trial information: NCT00079066. [Table: see text]

Comprehensive analysis of KRAS and NRAS mutations as predictive biomarkers for single agent panitumumab (pmab) response in a randomized, phase III metastatic colorectal cancer (mCRC) study (20020408).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3617-3617 ◽  
Author(s):  
Scott D. Patterson ◽  
Marc Peeters ◽  
Salvatore Siena ◽  
Eric Van Cutsem ◽  
Yves Humblet ◽  
...  
Keyword(s):  
Single Agent ◽  
Objective Response ◽  
Predictive Biomarkers ◽  
Progression Free Survival ◽  
Best Supportive Care ◽  
Phase Iii ◽  
Exon 2 ◽  
Biomarker Analysis ◽  
Exon 4 ◽  
Kras Exon

3617 Background: An exploratory biomarker analysis of the randomized, phase 3 monotherapy 20020408 study of pmab vs best supportive care (BSC) demonstrated that mutations in KRAS exon 3 and NRAS exons 2 and 3 appeared to be predictive of pmab response (Peeters et al, 2013). We expanded these results to determine whether mutations in exon 4 of the KRAS and NRAS genes are predictive for pmab treatment and to determine the treatment effect in the overall wild-type (WT) KRAS and NRAS population. Methods: Using a combination of Next Generation Sequencing, Sanger Sequencing, and WAVE-based SURVEYOR Scan Kits from Transgenomic, archival patient tumors were examined for mutations in KRAS and NRAS exon 4. These data were combined with previously presented data from KRAS and NRAS exon 2 and 3 analyses for evaluation of the comprehensive WT KRAS and NRAS subgroup. Results: 9/243 (3.7%) and 2/243 (0.8%) patient tumors with WT KRAS exon 2 status harbored a mutation in KRAS or NRAS exon 4, respectively. One tumor had mutations in both KRAS and NRAS exon 4. In the pmab arm, patients with WT KRAS and WT NRAS tumor status had an objective response rate (ORR) of 15% (11/72) whereas patients with mutant (MT) KRAS or MT NRAS tumor status had an ORR of 1% (1/95; 1 patient with MT KRAS exon 4 had a partial response). There were no responses in the BSC arm regardless of the tumor status. In this analysis set, the treatment hazard ratio (HR; pmab:BSC) for progression-free survival (PFS) in the WT KRAS and WT NRAS subgroup was 0.38 (95% CI: 0.27 - 0.56), and in the MT KRAS or MT NRAS subgroup was 0.98 (95% CI: 0.73 - 1.31). The original WT KRAS exon 2 subgroup PFS HR was 0.45 (95% CI: 0.34 - 0.59) (Amado et al, 2007). Conclusions: This exploratory analysis suggests that mutations in KRAS and NRAS exon 4 occur in a small, but meaningful percentage of patients with mCRC. Extending previous findings from this study in patients with MT KRAS and/or MT NRAS exon 2 and/or 3 tumors, patients with MT KRAS and/or MT NRAS exon 4 tumors do not appear to benefit from pmab therapy. Clinical trial information: NCT00113763.

Analysis of KRAS/NRAS, PI3CA, and BRAF mutations in the phase II KSCC0901 study of cetuximab plus S-1 as third-line treatment for metastatic colorectal cancer in Japanese patients.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 587-587
Author(s):  
Yasunori Emi ◽  
Eiji Oki ◽  
Hiroshi Saeki ◽  
Hiroyuki Kitao ◽  
Hironori Samura ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Treatment Protocol ◽  
Progression Free Survival ◽  
Japanese Patients ◽  
Phase Iii ◽  
Braf Mutations ◽  
Full Spectrum ◽  
Exon 2 ◽  
Third Line ◽  
Kras Exon

587 Background: Exploratory biomarker studies in the randomized phase III PRIME study suggested that patients with mCRC tumors harboring a mutation (MT) in KRAS or NRAS (beyond KRAS exon 2) do not respond to panitumumab-FOLFOX4 (2013 ASCO, JCO 31, #3511). We reported the efficacy and safety of S-1+. Cetuximab (Cmab) in wild-type (WT)-KRAS (exon 2) mCRC patients (pts) as third-line therapy (KSCC0901) (2012 ASCO, JCO 30, s3558). The full spectrum of characterized RAS (KRAS and NRAS), PI3CA, and BRAFMTs may affect efficacy in S-1+Cmab therapy in Japanese patients. Methods: An analysis was conducted to assess the treatment effect of S-1+Cmab, according to RAS (KRAS and NRAS), PI3CA, and BRAF MTs. The treatment protocol of KSCC0901 was as follows: weekly intravenous Cmab administration of 400 mg/m2 (day 1), 250 mg/m2/week (following day 1) and oral administration of 80 mg/m2/day S-1 on days 1–28 of each 42-day cycle. MTs in KRAS codons 12 and 13 (exon 2) were detected using direct sequencing according to the manufacturer’s instructions. MTs in KRAS codons 61 and 146, NRAS codons 12, 13 and 61, BRAF codon 600 and PIK3CA codons 542, 545, 546, and 1047 were detected using the multiplex PCR-Luminex method-based MEBGEN MT Kit (Medical and Biological Laboratories, Nagoya, Japan). Results: 35 pts were eligible for the analysis. Number of MT cases of KRAS, NRAS, BRAF, and PIK3CA were 4, 2, 2, and 5, respectively. One case had MTs of NRAS and PIK3CA. In 37 cases with WT KRASexon2 pts, median progression-free survival (mPFS) was 5.6 [95% CI: 4.4 – 5.7] months; median overall survival (mOS), 13.5 [8.5-16.5] months; and best overall response (ORR), 37.8%. In the all-WT subgroup 23 cases, mPFS was 5.6 [5.1-7.1] months; mOS, 13.5 [9.3-21.2] months; and best ORR, 47.8%. In 12 patients with any MT, mPFS was 4.0 [2.7-7.5] months; mOS, 5.5[2.9- ] months; and best ORR, 16.7%. Conclusions: These results suggest that also in Japanese patients receiving a Cmab containing regimen, RAS, BRAF and PIK3CA MT status beyond KRAS exon2 might predict non-responders to treatment. Clinical trial information: UMIN000002475.

Outcomes by hypomagnesemia (hypomag) in the randomized phase III ASPECCT trial of patients (pts) with chemofractory wild-type (WT) KRAS exon 2 metastatic colorectal cancer (mCRC).

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 507-507
Author(s):  
Marc Peeters ◽  
Tae Won Kim ◽  
Jin Li ◽  
Stefano Cascinu ◽  
Paul Ruff ◽  
...  
Keyword(s):  
Ad Hoc ◽  
Final Analysis ◽  
Phase Iii ◽  
Exon 2 ◽  
Trial Analysis ◽  
Grade 3 ◽  
Dose Modifications ◽  
To Receive ◽  
Kras Exon ◽  
Clinical Trial Information

507 Background: ASPECCT demonstrated that panitumumab (pmab) was noninferior to cetuximab (cmab) for overall survival (OS). Ad hoc analyses from ASPECCT suggested that hypomag was associated with better outcomes for pmab and cmab (Price 2015). However, results from the phase 3 NCIC CTG/AGITG CO.17 trial indicated hypomag at day 28 was associated with worse outcomes for cmab (Vickers 2013). Methods: Patients (pts) with chemorefractory WT KRASexon 2 mCRC were randomized 1:1 to receive pmab or cmab. Ad hoc analyses by hypomag were performed from the final analysis of ASPECCT at week 5, consistent with the NCIC CTG/AGITG CO.17 trial analysis (Vickers, 2013). Results: 999 pts were treated: 496 received pmab and 503 received cmab. Any grade hypomag was 29.0% and grade ≥3 was 7.3% in the pmab arm vs 19.3% and 2.8% in the cmab arm, respectively. In the pmab arm, 1.2% of pts discontinued treatment and 5% of pts had dose modifications due to hypomag vs 0.4% and 3% in the cmab arm, respectively. Efficacy results by hypomag are shown (Table). Conclusions: In ASPECCT, rates of hypomag were higher in the pmab vs the cmab arm. Pts who developed any grade hypomag with pmab or cmab had longer median OS compared with those pts who did not. Consistent with previous analyses, development of hypomag at week 5 was associated with worse median OS for cmab. Clinical trial information: NCT01001377. [Table: see text]

The prevalence RAS and BRAF mutations among patients in the Middle East and Northern Africa with metastatic colorectal cancer.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 598-598
Author(s):  
Tamer Garawin ◽  
Kimberly Lowe ◽  
George Kafatos ◽  
Samuel Murray
Keyword(s):  
Colorectal Cancer ◽  
Saudi Arabia ◽  
Middle East ◽  
Wild Type ◽  
Braf Mutations ◽  
Real World Data ◽  
Exon 2 ◽  
Ras Mutation ◽  
Exon 4 ◽  
Kras Exon

598 Background: Anti-EGFR therapies are recommended for metastatic colorectal cancer (mCRC) patients with confirmed wild-type RAS (exons 2, 3, 4 of KRAS and NRAS) status. There is limited published information on the prevalence of RAS mutations using real world data. The objective of this study was estimate the prevalence of RAS and BRAF mutations among patients with mCRC in the Middle East and Northern Africa (MENA) in an effort to inform the rationale for biomarker testing and treatment choice. Methods: The study included 1,669 patients from August 2013 to July 2015 with mCRC from Algeria, Bahrain, Egypt, Jordan, Kuwait, Lebanon, Libya, Morocco, Oman, Qatar, Saudi Arabia, and the United Arab Emeritus. Information on RAS mutation status was obtained from one pathology lab using High Resolution Melting Analysis. Extended RAS analysis was conducted in a subset of patients, including: overall RAS (exon 2, 3, 4 of KRAS and NRAS; n = 750), KRAS exon 2 (n = 750), KRAS exon 3 and 4 (n = 507), NRAS exon 2, 3, and 4 (n = 507), and BRAF exon 15 (n = 78). The proportion of patients with each mutation was summarized. Results: The overall RAS mutation in the full sample was 35.3% (n = 589/1669). The observed mutation for KRAS exon 2 in a subset of patients with extended RAS analysis (n = 750) was 32.4% (243/750). Out of the subjects with wild-type exon 2 (n = 507), the observed mutations rates were as follows: KRAS exon 4 (20/507 = 3.9%), KRAS exon 3 (13/507 = 2.6%), NRAS exon 2 (7/507 = 1.4%), NRAS exon 3 (6/507 = 1.2%), and NRAS exon 4 (0%). The prevalence of BRAF exon 15 was 3.8% (3/78). The most robust data on specific RAS mutations was obtained from Algeria, Egypt, and Saudi Arabia. The prevalence of KRAS exon 2 mutations in these countries was as follows: Algeria (n = 33/86 = 38.4%), Egypt (n = 83/303 = 27.4%), Saudi Arabia (n = 85/245 = 34.7%). Conclusions: To our knowledge, this is the first study to evaluate the prevalence of RAS and BRAF mutations in the Middle East using real world data. The results of this descriptive study illustrate that there is variation in the prevalence of RAS and BRAF mutations in MENA.

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

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3557-3557
Author(s):  
Robin Park ◽  
Laércio Lopes da Silva ◽  
Sunggon Lee ◽  
Anwaar Saeed
Keyword(s):  
Colorectal Cancer ◽  
Systematic Review ◽  
Braf Mutation ◽  
Meta Analysis ◽  
Stage Iv ◽  
Stage I ◽  
Prognostic Impact ◽  
Braf Mutations ◽  
Mutation Status ◽  
The Impact

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]

Quality Control and Telemedicine for BRAF V600E Mutations in Papillary Thyroid Carcinomas

2015 ◽  
Vol 4 (2) ◽  
pp. 12-30 ◽  
Author(s):  
Niki Margari ◽  
Abraham Pouliakis ◽  
Aris Spathis ◽  
Emmanouil Mastorakis ◽  
Efthymios Karakostas ◽  
...  
Keyword(s):  
Quality Control ◽  
Braf Mutation ◽  
Image Features ◽  
Classification And Regression Tree ◽  
Braf V600e ◽  
Papillary Thyroid ◽  
Cell Nuclei ◽  
Braf Mutations ◽  
Mutation Status ◽  
Thyroid Carcinomas

The assessment of BRAF V600E mutations is important for prognosis and treatment of Papillary Thyroid Carcinomas (PTC), the standard methods for their identification are molecular biology techniques. In this study, the potential of image morphometry applied to cell nuclei and sequentially the use of a Classification And Regression Tree (CART) is investigated, in order to: identify morphometric features useful to characterize BRAF mutations, and to eventually produce an algorithm identifying BRAF mutation status. The 140 studied cases had histological confirmation and known BRAF mutation status identified via real-time PCR. The analysis revealed that nuclear features contributing to BRAF mutation status identification via the CART model are related mostly to nuclear color. According to the results there is evidence that BRAF V600E mutations can be identified by measurable image features. Therefore, the proposed method is useful for quality control of BRAF V600E mutations on cytological slides, can serve as alternative to PCR method and may be used for remote assessment.

scholarly journals 7 Assessing melanoma BRAF status through ddPCR of cfDNA

2019 ◽  
Vol 95 (1130) ◽  
pp. 686.4-687
Author(s):  
Lauren Passy ◽  
Shobha Silva ◽  
Ian Brock ◽  
Greg Wells ◽  
Angela Cox ◽  
...  
Keyword(s):  
Braf Mutation ◽  
Limit Of Detection ◽  
Wild Type ◽  
Braf Mutations ◽  
Mutation Status ◽  
Gapdh Gene ◽  
Fractional Abundance ◽  
Significant Difference ◽  
Tissue Specimens ◽  
Braf Mutant

IntroductionTreatment of recurrent and metastatic melanoma has been revolutionised by targeted therapy. Inhibitors of mutant BRAF are a systemic treatment offered for patients with stage III/IV melanoma who are known to carry a mutation in BRAF. Currently patients’ BRAF mutation status is assessed through molecular analysis of tissue specimens.Cell-free DNA (cfDNA) released from tumours can be used to non-invasively detect active disease and predict survival in melanoma. cfDNA also provides a method for detecting BRAF mutations. This project aimed to ascertain BRAF mutation status in cfDNA through digital droplet PCR (ddPCR) of plasma samples from patients with melanoma. We aimed to assess the relationship between cfDNA BRAF positivity and disease relapse and progression.MethodsPlasma from 100 patients with active or recently resected melanoma was obtained during previous work. 85 samples had cfDNA extracted. Tissue BRAF status was known for 57 samples. cfDNA was extracted from 1–2 ml plasma with the QIAamp circulating nucleic acid kit (QIAGEN®) following manufacturer protocol, eluting cfDNA into 100µL. cfDNA was quantified with SYBR green quantitative real-time PCR (Life Technologies), based on an 87bp GAPDH gene amplicon. ddPCR™ was performed using the Bio-Rad QX200 Droplet Generator™ and Droplet Reader as per manufacturer protocol. Analysis was performed with Bio-Rad QuantaSoft Version 1.7.4.ResultsMedian yield of cfDNA extracted from 85 samples was 1.97 ng/ml when eluted into 100µL. This was well-correlated with previous cfDNA extraction yields from this sample set (Pearson’s r=0.6687, p<0.0005), where a 200µL elution volume was used. 74 samples yielded >10,000 droplets and were included for analysis. 12 samples contained BRAF mutant positive droplets. A 74% concordance rate between tissue BRAF mutation status and the presence/absence of cfDNA BRAF mutant positive droplets was found. 7/18 tissue BRAF mutant samples contained BRAF mutant droplets, in comparison to 2/32 tissue BRAF wild-type samples. The presence of BRAF mutant positive droplets was significantly different between the tissue BRAF mutant and tissue BRAF wild-type groups (χ2 8.3145, p=0.004).Fractional abundance of BRAF mutant droplets in the samples containing mutant droplets ranged from 0.07–0.74%. When comparing BRAF mutant droplet-containing samples and samples without BRAF mutant droplets, there was no significant difference in rate of relapse (χ2 0.0948, p=0.758), nor mortality rate (χ2 3.3959, p=0.654).Conclusion cfDNA provides a non-invasive snapshot of the tumour genome and any potential therapeutic targets held within. This work demonstrates that a very low volume of cfDNA can be used to detect BRAF mutations in patients with melanoma through ddPCR.Previous work assessing BRAF status in cfDNA has used larger volumes of cfDNA. Though our concordance rates are comparable with other studies, it is possible that using a smaller amount of cfDNA in our ddPCR has resulted in some samples being below the limit of detection for ddPCR.Longitudinal study is warranted to monitor cfDNA BRAF status and mutant fractional abundance, and whether this better correlates with relapse of disease and disease progression.

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