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Variations of BRAF mutant allele percentage in melanomas.

Hélias-Rodzewicz Z, Funck-Brentano E, Baudoux L, Jung CK, Zimmermann U, Marin C, Clerici T, Le Gall C, Peschaud F, Taly V, Saiag P, Emile JF - BMC Cancer (2015)

Bottom Line: FISH showed a polysomy of chromosome 7 in 13.6%, 35.3% and 54.5% of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005).Amplification (5.6%) and loss (3.2%) of BRAF locus were rare.By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi.

View Article: PubMed Central - PubMed

Affiliation: EA4340, Versailles University, Boulogne-Billancourt, France. zofia.helias@aphp.fr.

ABSTRACT

Background: BRAF mutations are present in 40% of human skin melanomas. Mutated tumors with an increased percentage of BRAF mutant alleles (BRAF-M%) may have a better response to RAF/MEK inhibitors. We evaluated the BRAF-M% in melanomas, and the genetic causes of its variation.

Methods: BRAF-M% was quantified by pyrosequencing, real-time PCR (rtPCR) and/or picoliter-droplet PCR (dPCR). BRAF mutant expression was detected by immunohistochemistry. Chromosomal alterations were analyzed with fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) arrays.

Results: BRAF-M% quantification obtained with pyrosequencing was highly correlated (R = 0.94) with rtPCR, and with dPCR. BRAF-M% quantified from DNA and RNA were also highly correlated (R = 0.98). Among 368 samples with >80% tumor cells, 38.6% had a BRAF (V600E) mutation. Only 66.2% cases were heterozygous (BRAF-M% 30 to 60%). Increased BRAF-M% (>60%) was observed in 19% of cases. FISH showed a polysomy of chromosome 7 in 13.6%, 35.3% and 54.5% of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005). Amplification (5.6%) and loss (3.2%) of BRAF locus were rare. By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi.

Conclusions: BRAF-M% is heterogeneous and frequently increased in BRAF-mutant melanomas. Aneuploidy of chromosome 7 is more frequent in BRAF mutant melanomas, specifically in those with high BRAF-M%.

No MeSH data available.


Related in: MedlinePlus

Variations in the percentage of BRAF V600E mutation in melanomas. a Scatter plot representation of the amounts of wild-type and V600E BRAF allele distribution in relation to the percentage of tumor cells in 475 FFPE melanoma samples. b Histogram representation of the percentage of BRAF V600E mutated allele in 368 melanomas obtained by pyrosequencing analysis. The Y axis corresponding to the number of cases is broken between 50 and 200
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Fig1: Variations in the percentage of BRAF V600E mutation in melanomas. a Scatter plot representation of the amounts of wild-type and V600E BRAF allele distribution in relation to the percentage of tumor cells in 475 FFPE melanoma samples. b Histogram representation of the percentage of BRAF V600E mutated allele in 368 melanomas obtained by pyrosequencing analysis. The Y axis corresponding to the number of cases is broken between 50 and 200

Mentions: Pyrosequencing quantification was obtained in 475 FFPE melanoma samples from 428 patients with AJCC stage III or IV melanoma with either V600E or WT BRAF, after histological evaluation of the percentage of tumor cells (flow chart in Additional file 4). No discordance concerning BRAF mutational status was observed among the 46 patients with at least two distinct melanoma samples, and the median BRAF-M% variation was 2.5 %. As expected, according to the percentage of tumor cells in mutated BRAF melanomas, we observed a distinct distribution of the percentage of mutated allele (Fig. 1a). The inter-pathologist reproducibility for the evaluation of tumor cell content was substantial for the 80 % cut-off (κ = 0.79), and was only moderate for the 70 % cut-off (κ = 0.49). Thus, we decided to use the cut-off of 80 % of tumor cells. The distribution of mutated BRAF amounts in samples with less (n = 107) or more (n = 368) than 80 % of tumor cells were significantly different (P < 0.05). Therefore, we thus excluded samples with less than 80 % of melanoma cells from further analysis.Fig. 1


Variations of BRAF mutant allele percentage in melanomas.

Hélias-Rodzewicz Z, Funck-Brentano E, Baudoux L, Jung CK, Zimmermann U, Marin C, Clerici T, Le Gall C, Peschaud F, Taly V, Saiag P, Emile JF - BMC Cancer (2015)

Variations in the percentage of BRAF V600E mutation in melanomas. a Scatter plot representation of the amounts of wild-type and V600E BRAF allele distribution in relation to the percentage of tumor cells in 475 FFPE melanoma samples. b Histogram representation of the percentage of BRAF V600E mutated allele in 368 melanomas obtained by pyrosequencing analysis. The Y axis corresponding to the number of cases is broken between 50 and 200
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491198&req=5

Fig1: Variations in the percentage of BRAF V600E mutation in melanomas. a Scatter plot representation of the amounts of wild-type and V600E BRAF allele distribution in relation to the percentage of tumor cells in 475 FFPE melanoma samples. b Histogram representation of the percentage of BRAF V600E mutated allele in 368 melanomas obtained by pyrosequencing analysis. The Y axis corresponding to the number of cases is broken between 50 and 200
Mentions: Pyrosequencing quantification was obtained in 475 FFPE melanoma samples from 428 patients with AJCC stage III or IV melanoma with either V600E or WT BRAF, after histological evaluation of the percentage of tumor cells (flow chart in Additional file 4). No discordance concerning BRAF mutational status was observed among the 46 patients with at least two distinct melanoma samples, and the median BRAF-M% variation was 2.5 %. As expected, according to the percentage of tumor cells in mutated BRAF melanomas, we observed a distinct distribution of the percentage of mutated allele (Fig. 1a). The inter-pathologist reproducibility for the evaluation of tumor cell content was substantial for the 80 % cut-off (κ = 0.79), and was only moderate for the 70 % cut-off (κ = 0.49). Thus, we decided to use the cut-off of 80 % of tumor cells. The distribution of mutated BRAF amounts in samples with less (n = 107) or more (n = 368) than 80 % of tumor cells were significantly different (P < 0.05). Therefore, we thus excluded samples with less than 80 % of melanoma cells from further analysis.Fig. 1

Bottom Line: FISH showed a polysomy of chromosome 7 in 13.6%, 35.3% and 54.5% of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005).Amplification (5.6%) and loss (3.2%) of BRAF locus were rare.By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi.

View Article: PubMed Central - PubMed

Affiliation: EA4340, Versailles University, Boulogne-Billancourt, France. zofia.helias@aphp.fr.

ABSTRACT

Background: BRAF mutations are present in 40% of human skin melanomas. Mutated tumors with an increased percentage of BRAF mutant alleles (BRAF-M%) may have a better response to RAF/MEK inhibitors. We evaluated the BRAF-M% in melanomas, and the genetic causes of its variation.

Methods: BRAF-M% was quantified by pyrosequencing, real-time PCR (rtPCR) and/or picoliter-droplet PCR (dPCR). BRAF mutant expression was detected by immunohistochemistry. Chromosomal alterations were analyzed with fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) arrays.

Results: BRAF-M% quantification obtained with pyrosequencing was highly correlated (R = 0.94) with rtPCR, and with dPCR. BRAF-M% quantified from DNA and RNA were also highly correlated (R = 0.98). Among 368 samples with >80% tumor cells, 38.6% had a BRAF (V600E) mutation. Only 66.2% cases were heterozygous (BRAF-M% 30 to 60%). Increased BRAF-M% (>60%) was observed in 19% of cases. FISH showed a polysomy of chromosome 7 in 13.6%, 35.3% and 54.5% of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005). Amplification (5.6%) and loss (3.2%) of BRAF locus were rare. By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi.

Conclusions: BRAF-M% is heterogeneous and frequently increased in BRAF-mutant melanomas. Aneuploidy of chromosome 7 is more frequent in BRAF mutant melanomas, specifically in those with high BRAF-M%.

No MeSH data available.


Related in: MedlinePlus