Limits...
BRAF and NRAS mutations are heterogeneous and not mutually exclusive in nodular melanoma.

Chiappetta C, Proietti I, Soccodato V, Puggioni C, Zaralli R, Pacini L, Porta N, Skroza N, Petrozza V, Potenza C, Della Rocca C, Di Cristofano C - Appl. Immunohistochem. Mol. Morphol. (2015)

Bottom Line: This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis.Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations.Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

View Article: PubMed Central - PubMed

Affiliation: *UOC of Pathology, Department of Medical-Surgical Sciences and Bio-Technologies, Sapienza University of Rome, Polo Pontino, I.C.O.T, Latina †UOC of Dermatology "Daniele Innocenzi," Department of Medical-Surgical Sciences and Bio-Technologies, Sapienza University of Rome, Fiorini Hospital, Polo Pontino, Terracina, Italy.

ABSTRACT
Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there's a subset of such patients who do not respond to the therapy. Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor. We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis. Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations. Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation. This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis. Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations. Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

Show MeSH

Related in: MedlinePlus

Sequencing electropherograms of the regions of BRAF exons 11 and 15 and NRAS exons 1 and 2. In each line, the first electropherogram corresponds to the tumoral sample, the other 3 electropherograms correspond to the 3 areas of the microdissected tumor. A, Patient that shows the same 2 mutations of tumor sample (S467L of BRAF exon 11, Q61R of NRAS exon 2) in all 3 areas; (B) patient that shows the same mutation found in tumoral sample in all 3 areas (Q61R of NRAS exon 2) and also other 2 mutation in 2 different areas (A598V and V600R on BRAF exon 15, respectively); (C) patient that shows the V600E mutation of BRAF exon 15 in all 3 areas while the tumoral sample is referred to as wild type (WT).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482453&req=5

Figure 1: Sequencing electropherograms of the regions of BRAF exons 11 and 15 and NRAS exons 1 and 2. In each line, the first electropherogram corresponds to the tumoral sample, the other 3 electropherograms correspond to the 3 areas of the microdissected tumor. A, Patient that shows the same 2 mutations of tumor sample (S467L of BRAF exon 11, Q61R of NRAS exon 2) in all 3 areas; (B) patient that shows the same mutation found in tumoral sample in all 3 areas (Q61R of NRAS exon 2) and also other 2 mutation in 2 different areas (A598V and V600R on BRAF exon 15, respectively); (C) patient that shows the V600E mutation of BRAF exon 15 in all 3 areas while the tumoral sample is referred to as wild type (WT).

Mentions: We analyzed the 3 microdissected areas of each tumor sample to determine whether they contained a mixture of subclones characterized by a different mutational status. We found that 7/11 (64%) mutated patients showed the same mutation found by mutational analysis without LCM in at least 1 area. Only 4 patients (57%, 4/7) showed the same mutation in all 3 areas, specifically V600E in exon 15 of BRAF in 2 patients and the glycine to arginine substitution at position 13 of NRAS exon 1 (G13R) in the third patient; the patient that showed 2 different mutations in the same tumor specimen revealed this characteristic through the LCM analysis in all 3 areas too (S467L BRAF exon 11 and Q61R NRAS exon 2, Fig. 1A). Two patients (29%, 2/7) showed the same mutation in 2 areas: the lysine to glutamic acid substitution at position 601 of BRAF exon 15 (K601E) and the other showed the substitution of valine in arginine in exon 15 of BRAF (V600R) while 1 area resulting WT. Only 1 patient (14%, 1/7) showed the same mutation in 1 area, the valine to lysine substitution at position 600 of BRAF exon 15 (V600K), whereas the other areas resulted WT. Four patients (36%, 4/11) revealed a different mutational status in at least 1 area compared with mutational analysis without LCM: 1 patient (25%, 1/4) showed the same mutation in 2 areas (V600E in exon 15 of BRAF) and another mutation (V600R) in the same exon in the third area; 2 patients (50%, 2/4) showed the same mutation in all 3 areas (Q61R and Q61K) and also other mutations in BRAF exon 15, in particular the first showed the V600R mutation and the alanine to valine substitution at position 598 (A598V, Fig. 1B) and the last showed the isoleucine to valine substitution at position 592 (I592V); another patient (25%, 1/4) showed the same mutation in 2 areas (Q61L) and also revealed the glycine to arginine substitution at position 455 of BRAF exon 11 (G455R). In the group referred to as WT, only 1 patient (1/4, 25%) showed a V600E mutation in all 3 areas (Fig. 1C). The last 3 patients (3/4, 75%) were also referred to as WT by LCM analysis.


BRAF and NRAS mutations are heterogeneous and not mutually exclusive in nodular melanoma.

Chiappetta C, Proietti I, Soccodato V, Puggioni C, Zaralli R, Pacini L, Porta N, Skroza N, Petrozza V, Potenza C, Della Rocca C, Di Cristofano C - Appl. Immunohistochem. Mol. Morphol. (2015)

Sequencing electropherograms of the regions of BRAF exons 11 and 15 and NRAS exons 1 and 2. In each line, the first electropherogram corresponds to the tumoral sample, the other 3 electropherograms correspond to the 3 areas of the microdissected tumor. A, Patient that shows the same 2 mutations of tumor sample (S467L of BRAF exon 11, Q61R of NRAS exon 2) in all 3 areas; (B) patient that shows the same mutation found in tumoral sample in all 3 areas (Q61R of NRAS exon 2) and also other 2 mutation in 2 different areas (A598V and V600R on BRAF exon 15, respectively); (C) patient that shows the V600E mutation of BRAF exon 15 in all 3 areas while the tumoral sample is referred to as wild type (WT).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482453&req=5

Figure 1: Sequencing electropherograms of the regions of BRAF exons 11 and 15 and NRAS exons 1 and 2. In each line, the first electropherogram corresponds to the tumoral sample, the other 3 electropherograms correspond to the 3 areas of the microdissected tumor. A, Patient that shows the same 2 mutations of tumor sample (S467L of BRAF exon 11, Q61R of NRAS exon 2) in all 3 areas; (B) patient that shows the same mutation found in tumoral sample in all 3 areas (Q61R of NRAS exon 2) and also other 2 mutation in 2 different areas (A598V and V600R on BRAF exon 15, respectively); (C) patient that shows the V600E mutation of BRAF exon 15 in all 3 areas while the tumoral sample is referred to as wild type (WT).
Mentions: We analyzed the 3 microdissected areas of each tumor sample to determine whether they contained a mixture of subclones characterized by a different mutational status. We found that 7/11 (64%) mutated patients showed the same mutation found by mutational analysis without LCM in at least 1 area. Only 4 patients (57%, 4/7) showed the same mutation in all 3 areas, specifically V600E in exon 15 of BRAF in 2 patients and the glycine to arginine substitution at position 13 of NRAS exon 1 (G13R) in the third patient; the patient that showed 2 different mutations in the same tumor specimen revealed this characteristic through the LCM analysis in all 3 areas too (S467L BRAF exon 11 and Q61R NRAS exon 2, Fig. 1A). Two patients (29%, 2/7) showed the same mutation in 2 areas: the lysine to glutamic acid substitution at position 601 of BRAF exon 15 (K601E) and the other showed the substitution of valine in arginine in exon 15 of BRAF (V600R) while 1 area resulting WT. Only 1 patient (14%, 1/7) showed the same mutation in 1 area, the valine to lysine substitution at position 600 of BRAF exon 15 (V600K), whereas the other areas resulted WT. Four patients (36%, 4/11) revealed a different mutational status in at least 1 area compared with mutational analysis without LCM: 1 patient (25%, 1/4) showed the same mutation in 2 areas (V600E in exon 15 of BRAF) and another mutation (V600R) in the same exon in the third area; 2 patients (50%, 2/4) showed the same mutation in all 3 areas (Q61R and Q61K) and also other mutations in BRAF exon 15, in particular the first showed the V600R mutation and the alanine to valine substitution at position 598 (A598V, Fig. 1B) and the last showed the isoleucine to valine substitution at position 592 (I592V); another patient (25%, 1/4) showed the same mutation in 2 areas (Q61L) and also revealed the glycine to arginine substitution at position 455 of BRAF exon 11 (G455R). In the group referred to as WT, only 1 patient (1/4, 25%) showed a V600E mutation in all 3 areas (Fig. 1C). The last 3 patients (3/4, 75%) were also referred to as WT by LCM analysis.

Bottom Line: This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis.Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations.Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

View Article: PubMed Central - PubMed

Affiliation: *UOC of Pathology, Department of Medical-Surgical Sciences and Bio-Technologies, Sapienza University of Rome, Polo Pontino, I.C.O.T, Latina †UOC of Dermatology "Daniele Innocenzi," Department of Medical-Surgical Sciences and Bio-Technologies, Sapienza University of Rome, Fiorini Hospital, Polo Pontino, Terracina, Italy.

ABSTRACT
Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there's a subset of such patients who do not respond to the therapy. Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor. We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis. Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations. Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation. This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis. Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations. Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

Show MeSH
Related in: MedlinePlus