Limits...
NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

Uguen A, Talagas M, Costa S, Samaison L, Paule L, Alavi Z, De Braekeleer M, Le Marechal C, Marcorelles P - Diagn Pathol (2015)

Bottom Line: Proportions and intensities of staining were varied.Technical limitations are discussed to explain those discrepancies.Anyway we could not rule out real tumor heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U1078, Brest, F-29200, France. arnaud.uguen@chu-brest.fr.

ABSTRACT

Background: The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies in the theranostic mutation screening of melanomas.

Methods: 142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry.

Results: 23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS (Q61R) and 23 BRAF (V600E) -mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS (Q61L) , NRAS (Q61K) , BRAF (V600K) and BRAF (V600R) mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity.

Conclusions: In our study, we showed that combining immunohistochemistry analysis targeting NRAS(Q61R) and BRAF(V600E) proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.

No MeSH data available.


Related in: MedlinePlus

Examples of paired immunohistochemistry and pyrosequencing results. a, b, c, d case #15: primary BRAFV600 wild-type (a) and NRASQ61R mutated (b) melanoma with strong 4+ immunostaining with SP 174 anti-NRASQ61Rantibody using Red (c) or DAB (d) revelation. e, f, g, h case #18: primary melanoma with BRAFV600 (e) and NRASQ61 wild-type (f) molecular status but presenting strong 4+ staining using VE1 anti-BRAFV600E antibody (g) and no staining with SP 174 anti-NRASQ61R antibody (h). i, j, k, l case #61: primary BRAFV600 wild-type (i) and NRASQ61K mutated (j) melanoma having moderate staining using VE1 anti-BRAFV600E antibody (k) and fainter staining with SP 174 anti-NRASQ61Rantibody (l). We concluded in a non-specific ambiguous staining in this sample with both antibodies. Note that red revelation kit as been used here as the faint melanin-pigmentation could simulate a weak DAB staining. m, n, o, p case #39: BRAFV600 (m) and NRASQ61 wild-type (n) melanoma mesentery metastasis presenting both strong melanin pigment that could simulate a strong DAB staining and red immunostained cells with both VE1 anti-BRAFV600E (o) and SP 174 anti-NRASQ61R (p) antibodies. We retrospectively concluded that all stained cells were macrophages, without any evidence of viable tumor cells in this pigmented sample. q, r case #55: BRAFV600E mutated (q) melanoma skin metastasis with strong 4+ immunostaining with VE1 anti-BRAFV600Eantibody (r)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4513673&req=5

Fig1: Examples of paired immunohistochemistry and pyrosequencing results. a, b, c, d case #15: primary BRAFV600 wild-type (a) and NRASQ61R mutated (b) melanoma with strong 4+ immunostaining with SP 174 anti-NRASQ61Rantibody using Red (c) or DAB (d) revelation. e, f, g, h case #18: primary melanoma with BRAFV600 (e) and NRASQ61 wild-type (f) molecular status but presenting strong 4+ staining using VE1 anti-BRAFV600E antibody (g) and no staining with SP 174 anti-NRASQ61R antibody (h). i, j, k, l case #61: primary BRAFV600 wild-type (i) and NRASQ61K mutated (j) melanoma having moderate staining using VE1 anti-BRAFV600E antibody (k) and fainter staining with SP 174 anti-NRASQ61Rantibody (l). We concluded in a non-specific ambiguous staining in this sample with both antibodies. Note that red revelation kit as been used here as the faint melanin-pigmentation could simulate a weak DAB staining. m, n, o, p case #39: BRAFV600 (m) and NRASQ61 wild-type (n) melanoma mesentery metastasis presenting both strong melanin pigment that could simulate a strong DAB staining and red immunostained cells with both VE1 anti-BRAFV600E (o) and SP 174 anti-NRASQ61R (p) antibodies. We retrospectively concluded that all stained cells were macrophages, without any evidence of viable tumor cells in this pigmented sample. q, r case #55: BRAFV600E mutated (q) melanoma skin metastasis with strong 4+ immunostaining with VE1 anti-BRAFV600Eantibody (r)

Mentions: All the 23 NRASQ61R-mutant samples detected by pyrosequencing, had positive immunostaining, with the NRASQ61R SP174 antibody (from 1+ to 4+, and from weak to strong) (see Table 3 and Fig. 1a–d. There was one NRASQ61R-wild-type sample who had positive immunostaining (2+, weak). The 7 NRASQ61L-mutant and the 8 NRASQ61K-mutant detected by pyrosequencing, were negative for immunostaining, with the NRASQ61R SP174 antibody.Fig. 1


NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

Uguen A, Talagas M, Costa S, Samaison L, Paule L, Alavi Z, De Braekeleer M, Le Marechal C, Marcorelles P - Diagn Pathol (2015)

Examples of paired immunohistochemistry and pyrosequencing results. a, b, c, d case #15: primary BRAFV600 wild-type (a) and NRASQ61R mutated (b) melanoma with strong 4+ immunostaining with SP 174 anti-NRASQ61Rantibody using Red (c) or DAB (d) revelation. e, f, g, h case #18: primary melanoma with BRAFV600 (e) and NRASQ61 wild-type (f) molecular status but presenting strong 4+ staining using VE1 anti-BRAFV600E antibody (g) and no staining with SP 174 anti-NRASQ61R antibody (h). i, j, k, l case #61: primary BRAFV600 wild-type (i) and NRASQ61K mutated (j) melanoma having moderate staining using VE1 anti-BRAFV600E antibody (k) and fainter staining with SP 174 anti-NRASQ61Rantibody (l). We concluded in a non-specific ambiguous staining in this sample with both antibodies. Note that red revelation kit as been used here as the faint melanin-pigmentation could simulate a weak DAB staining. m, n, o, p case #39: BRAFV600 (m) and NRASQ61 wild-type (n) melanoma mesentery metastasis presenting both strong melanin pigment that could simulate a strong DAB staining and red immunostained cells with both VE1 anti-BRAFV600E (o) and SP 174 anti-NRASQ61R (p) antibodies. We retrospectively concluded that all stained cells were macrophages, without any evidence of viable tumor cells in this pigmented sample. q, r case #55: BRAFV600E mutated (q) melanoma skin metastasis with strong 4+ immunostaining with VE1 anti-BRAFV600Eantibody (r)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4513673&req=5

Fig1: Examples of paired immunohistochemistry and pyrosequencing results. a, b, c, d case #15: primary BRAFV600 wild-type (a) and NRASQ61R mutated (b) melanoma with strong 4+ immunostaining with SP 174 anti-NRASQ61Rantibody using Red (c) or DAB (d) revelation. e, f, g, h case #18: primary melanoma with BRAFV600 (e) and NRASQ61 wild-type (f) molecular status but presenting strong 4+ staining using VE1 anti-BRAFV600E antibody (g) and no staining with SP 174 anti-NRASQ61R antibody (h). i, j, k, l case #61: primary BRAFV600 wild-type (i) and NRASQ61K mutated (j) melanoma having moderate staining using VE1 anti-BRAFV600E antibody (k) and fainter staining with SP 174 anti-NRASQ61Rantibody (l). We concluded in a non-specific ambiguous staining in this sample with both antibodies. Note that red revelation kit as been used here as the faint melanin-pigmentation could simulate a weak DAB staining. m, n, o, p case #39: BRAFV600 (m) and NRASQ61 wild-type (n) melanoma mesentery metastasis presenting both strong melanin pigment that could simulate a strong DAB staining and red immunostained cells with both VE1 anti-BRAFV600E (o) and SP 174 anti-NRASQ61R (p) antibodies. We retrospectively concluded that all stained cells were macrophages, without any evidence of viable tumor cells in this pigmented sample. q, r case #55: BRAFV600E mutated (q) melanoma skin metastasis with strong 4+ immunostaining with VE1 anti-BRAFV600Eantibody (r)
Mentions: All the 23 NRASQ61R-mutant samples detected by pyrosequencing, had positive immunostaining, with the NRASQ61R SP174 antibody (from 1+ to 4+, and from weak to strong) (see Table 3 and Fig. 1a–d. There was one NRASQ61R-wild-type sample who had positive immunostaining (2+, weak). The 7 NRASQ61L-mutant and the 8 NRASQ61K-mutant detected by pyrosequencing, were negative for immunostaining, with the NRASQ61R SP174 antibody.Fig. 1

Bottom Line: Proportions and intensities of staining were varied.Technical limitations are discussed to explain those discrepancies.Anyway we could not rule out real tumor heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U1078, Brest, F-29200, France. arnaud.uguen@chu-brest.fr.

ABSTRACT

Background: The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies in the theranostic mutation screening of melanomas.

Methods: 142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry.

Results: 23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS (Q61R) and 23 BRAF (V600E) -mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS (Q61L) , NRAS (Q61K) , BRAF (V600K) and BRAF (V600R) mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity.

Conclusions: In our study, we showed that combining immunohistochemistry analysis targeting NRAS(Q61R) and BRAF(V600E) proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.

No MeSH data available.


Related in: MedlinePlus