NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis - PubMed (original) (raw)

. 2014 Oct;134(10):2630-2638.

doi: 10.1038/jid.2014.154. Epub 2014 Mar 24.

Karin J Purdie 2, Stephen A Watt 1, Sam Haldenby 3, Nicoline den Breems 1, Michelle Dimon 4, Sarah T Arron 4, Michael J Kluk 5, Jon C Aster 5, Angela McHugh 1, Dylan J Xue 1, Jasbani Hs Dayal 1, Kim S Robinson 1, Sm Hasan Rizvi 6, Charlotte M Proby # 1, Catherine A Harwood # 2, Irene M Leigh # 1 2

Affiliations

NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis

Andrew P South et al. J Invest Dermatol. 2014 Oct.

Abstract

Cutaneous SCC (cSCC) is the most frequently occuring skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors. Here, we use massively parallel exome and targeted level sequencing of 132 sporadic cSCCs and of 39 squamoproliferative lesions and cSCCs arising in patients receiving the BRAF inhibitor vemurafenib, as well as 10 normal skin samples, to identify NOTCH1 mutation as an early event in squamous cell carcinogenesis. Bisected vemurafenib-induced lesions revealed surprising heterogeneity with different activating HRAS and NOTCH1 mutations identified in two halves of the same cSCC, suggesting polyclonal origin. Immunohistochemical analysis using an antibody specific to nuclear NOTCH1 correlates with mutation status in sporadic cSCCs, and regions of NOTCH1 loss or downregulation are frequently observed in normal-looking skin. Our data indicate that NOTCH1 acts as a gatekeeper in human cSCC.

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Figures

Figure 1

Figure 1. High mutation rate and spectra indicative of ultraviolet radiation damage in 20 sporadic cSCC samples

Upper bar graph shows exonic mutations per megabase for each individual tumor. Middle bar graph shows the spectrum of individual tumor somatic mutations as a percentage. Lower matrix depicts mutations (indicated by a filled square) in each individual tumor in genes previously reported to be frequently mutated in cSCC. Where more than one mutation is present in a single gene only one mutation type is given in the order delineated by color coded key; activating-Ras, nonsense, frameshift, splice site, missense.

Figure 2

Figure 2. 454 resequencing identifies prevalent mutation NOTCH1, NOTCH2 and TP53 in cSCC

Upper Matrix depicts mutations in the given gene for each individual tumor sample. Where more than one mutation is present in a single gene two mutations are shown in the order delineated by key; activating RAS, frameshift, nonsense, splice site, missense. Lower left matrix details mutations in 21 keratinocyte cSCC cell lines and lower right matrix details mutations in 39 skin lesions excised from 7 patients receiving the BRAF inhibitor vemurafenib. Mutations identified in normal skin DNA isolated from 6 of these 7 patients are shown to the right of this matrix.

Figure 3

Figure 3. NOTCH1 coverage is lower in exome capture compared with PCR amplification and 454 sequencing

A: Percentage of the NOTCH1 gene represented by 1X, 10X, 20X and 30X coverage in the first two samples sequenced using PCR amplification and 454 pyro-sequencing (454: TGM1 and 454: TGM2) compared with exome capture (Ex: WD_01 and Ex: WD_02). B: The mean allelic frequency of non-sense and missense mutations identified in NOTCH1 from 91 cSCC fresh frozen samples. Error shows standard deviation. Student T-Test p-value = 7e-06.

Figure 4

Figure 4. Bisected vemurafenib induced squamoproliferative lesions and cSCC display significant heterogeneity

Cartoons depict 8 bisected vemurafenib induced lesions and the resulting mutations identified in NOTCH1, NOTCH2, HRAS and TP53 from the isolated DNA. Solid lines enclose mutations in HRAS shared by both halves of the sample whilst dashed lines enclose different mutations in the same gene identified in the two halves of the sample. % indicates allelic frequency.

Figure 5

Figure 5. NOTCH1 Western blotting and immunohistochemistry correlates with mutation status and identifies regions of normal looking skin negative for NOTCH1 signaling

Standard 4-micron paraffin embedded tissue sections were stained using the Ventana Benchmark XT platform (Ventana Medical Systems, Inc., Tucson, AZ.) with extended heat induced epitope retrieval (CC1 Buffer). Slides were incubated for 1hr at room temperature with anti-NICD1 rabbit monoclonal antibody (clone D3B8, catalog #4147, Cell Signaling Technology, Beverly, MA; final concentration, 8.5microgram/mL). Signals were then amplified (Ventana Amplification Kit, #760-080) and visualized (Ventana Ultraview Universal DAB detection kit, #760-500) per the manufacturer’s instructions. A: Summary of immune-reactivity of 36 cSCC samples with anti-NICD1 antibody. B: Western blotting with 50μg of total cell lysate from SCCIC12 (no mutations), SCCT2 (missense, loss-of-function mutations) and SCCIC8 (homozygous PTC mutation) were resolved by 4-12% SDS-PAGE using standard techniques. The proteins were then transferred to a nitrocellulose membrane and visualized using antibodies recognizing full length NOTCH1, NICD1 and GAPDH. C: Immunohistochemistry data from sample TGP8, no NOTCH1 mutations. D: Sample TGW29, 100% STOP mutation. E: Sample TGM4, P391S 55%, G4247D 22%, P1860L 37% mutations. Left panel shows weakly positive region of the tumor while right panel shows negative region of the same tumor. F: Normal looking peri-tumor skin showing regions negative for NOTCH1 signaling adjacent to regions displaying the expected normal distribution of activated NOTCH1. Right panel shows extent of NOTCH1 negative patch flanked by positive NOTCH1 skin. All bars C-F = 300μm.

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