Improved Detection of the BRAF c.1799T>A (p.V600E) Mutation in Melanoma with a Single Nucleotide Primer Extension AssayGabriel C. Caponetti MD, Emilian Racila MD, Aaron Stence CLS, Jonathan Pruessner BS MT(ASCP),

Susan Forde CLS, Jason Hackman CLS, Deqin Ma MD, PhD, Jonathan Heusel MD, PhD and Aaron Bossler MD, PhD

University of Iowa Hospitals and Clinics, Department of Pathology, Iowa City, Iowa, USA

• BRAF mutations have been identified in ~66% of melanomas and in a smaller percentage of other tumors.

• The most common BRAF mutation is the c.1799T>A on exon 15 with the resultant p.V600E.

• Melanoma patients bearing this mutation can significantly benefit from vemurafenib therapy.

• Sanger sequencing (SS) is commonly used for mutation detection. However, its analytical sensitivity is limited at ~20%.

• Furthermore, melanin in high concentrations may inhibit Taq polymerase activity.

• Therefore, it is crucial to identify a sensitive method for mutation detection.

ResultsResults• The study group included melanoma cases from 10 females and 17 males with

ages ranging from 17 to 80 years (mean = 56.7 years). • The analytical sensitivity of the SNPE assay was as low as 2% (Fig 1). • In one case (case #1), the SNPE assay detected the mutation whereas the SS

did not. • In 22% (n=6) of the cases, the SS reaction initially failed but the SNPE assay

was able to assess for the presence or absence of the mutation. Only one SNPE reaction initially failed.

• In cases with initial failure of the SS reaction, the average melanin content was 39% (ranging from 1% to 90%) (Table 1).

• A significantly lower melanin content (0% to 40%, average 14%) was seen in cases with successful SS.

• By diluting the DNA (1:6 dilution with DNAse-free distilled water) and/or re-sampling of an area of tumor with less melanin (Fig. 2), we were able to successfully perform SS in most of the cases that had initially failed.

• One case correctly identified as a c.1798-1799GT>AA, V600K mutation by SS was detected by SNPE but identified as the c.1799T>A mutation.

BackgroundBackground

ConclusionConclusion• Although the number of cases analyzed in this series is small, our findings

suggest that the prevalence of the BRAF c.1799T>A (p.V600E) mutation in melanoma can be underestimated due the intrinsic limitations of SS and its susceptibility to interference by melanin.

• The SNPE assay is a much more sensitive alternative for the identification of this mutation and less susceptible to interference by melanin, which is a known inhibitor of PCR.

ReferencesReferences

Background: BRAF mutations have been identified in approximately 66% of melanomas. The most common BRAF mutation is the c.1799T>A on exon 15 with the resultant p.V600E. Melanoma patients bearing this mutation can significantly benefit from vemurafenib therapy. Sanger sequencing (SS) is the most commonly used test for the detection of this mutation. However, its analytical sensitivity is only 20% in formalin-fixed paraffin-embedded (FFPE) tissue. Therefore, it is crucial to identify a more sensitive method. In addition, melanin is known to inhibit Taq polymerase activity. The aims of this study were to determine the analytical sensitivity of a Single Nucleotide Primer Extension (SNPE) assay for the detection of the BRAF c.1799T>A mutation in melanoma and assess the impact of endogenous melanin on the performance of both assays.Design: Genomic DNA (gDNA) was extracted from FFPE tissue of 27 melanoma cases. The SNPE assay was performed using the ABI PRISM® SNaPshot™ kit (Applied Biosystems, Carlsbad, CA) in parallel with bi-directional SS. The analytical sensitivity of the SNPE assay was determined using purified plasmids containing the BRAF c.1799T>A (p.V600E) mutation at limiting dilutions admixed with 25 ng of wild type gDNA. The percentage of melanin content in the cases of melanoma was assessed by histological review.Results: The analytical sensitivity of the SNPE assay was as low as 2%. In 22% (n=6) of the cases, the SS reaction initially failed but the SNPE was able to assess for the presence or absence of the mutation. Only one SNPE reaction failed initially. In cases with initial failure of the SS reaction, the average melanin content was 39% (ranging from 1% to 90%). A significantly lower melanin content (0% to 40%, average 14%) was seen in cases with initial successful SS. By diluting the DNA or re-sampling of an area of tumor with less melanin, we were able to successfully perform SS in most of the cases that had initially failed.Conclusion: Although the number of cases analyzed in this series is small, our findings suggest that the prevalence of the BRAF c.1799T>A (p.V600E) mutation in melanoma can be underestimated due the intrinsic limitations of SS and its susceptibility to interference by melanin. The SNPE assay is a much more sensitive alternative for the identification of this mutation and less susceptible to interference by melanin. The development of a multiplexed SNPE assay for the identification of other BRAF mutations appears promising and is currently being evaluated.

The authors have no potential conflicts of interest to disclose.

AbstractAbstract Methods & MaterialsMethods & Materials• Bi-directional SS (Fig. 3) was performed in parallel with a SNPE assay. The

latter was performed using the ABI PRISM ® SNaPshot™ kit (Applied Biosystems, Carlsbad, CA).

• The analytical sensitivity of the SNPE assay was determined using purified plasmids containing the BRAF c.1799T>A (p.V600E) mutation at limiting dilutions admixed with 25 ng of wild type gDNA (Fig. 1).

• Genomic DNA (gDNA) was extracted from formalin-fixed paraffin-embedded tissue of 27 melanoma cases.

• The percentage of melanin content in the cases of melanoma was assessed by histological review (Fig. 2).

• 150ng (at 30ng/uL) and 25ng (at 25ng/uL) of tumoral DNA were used for the initial PCR for the SS and SNPE assays, respectively.

ObjectivesObjectives• To determine the analytical sensitivity of a Single Nucleotide Primer Extension

(SNPE) assay for the detection of the BRAF c.1799T>A mutation in melanoma and assess the impact of endogenous melanin on the performance of SS and the SNPE assay.

Case # Sanger SequencingSingle Nucleotide Primer

Extension assay for c.1799T>A, V600E

Total melanin content

Age / Gender

1 - c.1799T>A 1% 64, female2 c.1798-1799GT>AA c.1799T>A 20% 73, male3 c.1799T>A* c.1799T>A 80% 60, female4 - - 25% 72, male5 -* - 20% 53, male6 No sequencing result* c.1799T>A 30% 64, male7 - - 10% 80, female8 -* - 1% 54, female9 - - 5% 32, male

10 c.1799T>A* c.1799T>A 20% 57, male11 - - 0% 56, female12 - - 40% 36, female13 - - 5% 57, female14 c.1799T>A c.1799T>A 20% 32, female15 c.1799T>A c.1799T>A 10% 69, male16 - - 0% 74, male17 No sequencing result* c.1799T>A 30% 63, male18 - - 10% 57, male19 - - 25% 39, male20 - - 30% 53, male21 c.1799T>A* c.1799T>A* 90% 61, female 22 c.1799T>A c.1799T>A 10% 60, male23 - - 5% 67, male24 c.1799T>A c.1799T>A 0% 17, male25 - - 0% 70, male26 - - 20% 53, female27 - - 40% 59, male

Table 1. Comparison of BRAF mutations analysis on 27 cases of melanoma by SS and SNPE assay. A subset of results were discrepant due to melanin interference which affected primarily SS. In addition, in one case (case #1), the discrepancy in the results of both assays was due to the increased analytical sensitivity of the SNPE assay. *= the assay had to be repeated at least once, with dilution of the DNA, and/or re-sampling of an area of tumor with less melanin.

Figure 1. Composite image of BRAF mutation analysis results using SNPE showing the negative control and serial concentrations (2%, 5% and 50%) of plasmids with the BRAF V600E mutation

Figure 3. BRAF mutation analysis by SS (case # 22)

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Figure 2. Lymph node with metastatic melanoma (case # 3, H&E, 1X) with focally prominent melanin content (inset B, 40X). Inset A: section of tumor used for analysis (40X).c.1799T>A

c.1799T>A

Figure 4. BRAF mutation analysis by SNPE assay (case #22)

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