PMC7367153_01
Melanoma histology in Patient 1 (PT#1) and Patient 2 (PT#2) at different sites. Sections of PT#1 and PT#2 melanomas are from different melanoma sites as indicated and stained for haematoxilin and eosin. Magnification: 200x; sk, skin; ov, ovary; br, brain.
fonc-10-01056-g0001.jpg
PMC7367153_02_fonc-10-01056-g0001.jpg
F1
A 26-years-old female presented at clinic with a diagnosis of thick nodular melanoma of the skin trunk (from here referred as Pt#2sk) (Breslow thickness of 5.2 mm, ulcerated) (Figure 1), during her first pregnancy. At week 31, a cesarean section combined with right ovariectomy (from here referred as Pt#2 v) and a brain (from here referred as Pt#2br) symptomatic metastasectomy were carried out (Figure 1).
BRAF status in PT#1 and PT#2. Sections are from PT#1 and PT#2 and stained for anti-VE1 as labeled. Primary (PT#1 and PT#2sk) and metastatic (PT#2 v and PT#2br) melanomas are illustrated. A granular cytoplasmic stain for anti-BRAFV600E was detected in melanoma cells from all samples; no reactivity is observed in endothelial cells an immune cells. Sections were counterstained with hematoxylin. Magnification: 400x. Sequencing data illustrate the BRAF substitutions (Igv screenshot of amplicon that cover codon 600 and 601 of BRAF) at codon V600 and K601 in PT#1 and PT#2 by using Ion S5 system (PT#1) and Illumina MiSeq (PT#2sk, PT#2ov, and PT#2br).
fonc-10-01056-g0002.jpg
PMC7367153_02_fonc-10-01056-g0002.jpg
F2
Spectometry and Pyrosequencing, suggested a complex BRAF mutation involving codon V600 and K601 (not shown). Sanger sequencing of the BRAF exon 15 and the NGS-cancer panel confirmed the presence of the same three base pair substitution at the genomic level c.[1799_1800delinsAA; c.1802A>T] found in Pt#1 (Figure 2 and not shown) and leading to the p.V600E2; K601I change. Timeline with relevant data is included in Figure S2.
PTEN status in PT#1 and PT#2. Sections are from PT#1 and PT#2 and stained for anti-PTEN as labeled. Primary (PT#1 and PT#2sk) and metastatic (PT#2 v and PT#2br) melanomas are illustrated. Loss of PTEN reactivity was observed in melanoma cells from all samples except PT#2sk; internal positive control are represented by vessel. Sections were counterstained with hematoxylin and imaged at 100x magnification. Sequencing data illustrate the PTEN sequencing analysis (Igv screenshot of amplicon that cover exon 2 of PTEN) by Illumina MiSeq in P#T1 and PT#2 samples. A PTEN substitution is observed in samples PT#2ov and PT#2br. sk, skin; ov, ovary; br, brain.
fonc-10-01056-g0003.jpg
PMC7367153_02_fonc-10-01056-g0003.jpg
F3
Moreover, the NGS-cancer panel could also identify a pathogenic PTEN substitution (c.165-2A>G) in Pt#2ov and Pt#2br (Figure 3), leading to complete lack of PTEN expression by immunohistochemistry (Figure 3). Of note, melanoma cells from both cases co-stained for anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Figure S3).
PMC7367153_02
Melanoma histology in Patient 1 (PT#1) and Patient 2 (PT#2) at different sites. Sections of PT#1 and PT#2 melanomas are from different melanoma sites as indicated and stained for haematoxilin and eosin. Magnification: 200x; sk, skin; ov, ovary; br, brain.
fonc-10-01056-g0001.jpg
PMC7367153_02_fonc-10-01056-g0001.jpg
F1
A 26-years-old female presented at clinic with a diagnosis of thick nodular melanoma of the skin trunk (from here referred as Pt#2sk) (Breslow thickness of 5.2 mm, ulcerated) (Figure 1), during her first pregnancy. At week 31, a cesarean section combined with right ovariectomy (from here referred as Pt#2 v) and a brain (from here referred as Pt#2br) symptomatic metastasectomy were carried out (Figure 1).
BRAF status in PT#1 and PT#2. Sections are from PT#1 and PT#2 and stained for anti-VE1 as labeled. Primary (PT#1 and PT#2sk) and metastatic (PT#2 v and PT#2br) melanomas are illustrated. A granular cytoplasmic stain for anti-BRAFV600E was detected in melanoma cells from all samples; no reactivity is observed in endothelial cells an immune cells. Sections were counterstained with hematoxylin. Magnification: 400x. Sequencing data illustrate the BRAF substitutions (Igv screenshot of amplicon that cover codon 600 and 601 of BRAF) at codon V600 and K601 in PT#1 and PT#2 by using Ion S5 system (PT#1) and Illumina MiSeq (PT#2sk, PT#2ov, and PT#2br).
fonc-10-01056-g0002.jpg
PMC7367153_02_fonc-10-01056-g0002.jpg
F2
Spectometry and Pyrosequencing, suggested a complex BRAF mutation involving codon V600 and K601 (not shown). Sanger sequencing of the BRAF exon 15 and the NGS-cancer panel confirmed the presence of the same three base pair substitution at the genomic level c.[1799_1800delinsAA; c.1802A>T] found in Pt#1 (Figure 2 and not shown) and leading to the p.V600E2; K601I change. Timeline with relevant data is included in Figure S2.
PTEN status in PT#1 and PT#2. Sections are from PT#1 and PT#2 and stained for anti-PTEN as labeled. Primary (PT#1 and PT#2sk) and metastatic (PT#2 v and PT#2br) melanomas are illustrated. Loss of PTEN reactivity was observed in melanoma cells from all samples except PT#2sk; internal positive control are represented by vessel. Sections were counterstained with hematoxylin and imaged at 100x magnification. Sequencing data illustrate the PTEN sequencing analysis (Igv screenshot of amplicon that cover exon 2 of PTEN) by Illumina MiSeq in P#T1 and PT#2 samples. A PTEN substitution is observed in samples PT#2ov and PT#2br. sk, skin; ov, ovary; br, brain.
fonc-10-01056-g0003.jpg
PMC7367153_02_fonc-10-01056-g0003.jpg
F3
Moreover, the NGS-cancer panel could also identify a pathogenic PTEN substitution (c.165-2A>G) in Pt#2ov and Pt#2br (Figure 3), leading to complete lack of PTEN expression by immunohistochemistry (Figure 3). Of note, melanoma cells from both cases co-stained for anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Figure S3).