A novel germline NF1 splicing variant drives the onset of an anorectal mucosal melanoma in a patient with a stable and durable nivolumab response

Abstract

Objective: Neurofibromatosis type-1 (NF1) patients rarely develop mucosal melanomas. We report a rare form of anorectal mucosal melanoma (ARMM) in an NF1 syndromic patient profiled for genomics and transcriptomics to assess the determinants of the response to nivolumab.

Methods: Primary melanoma and metastases were analyzed with targeted sequencing and gene expression profile (tGEP). We applied in silico (cBioPortal and predictor tools) and in vitro (hybrid minigene) approaches to confirm the variant pathogenicity.

Results: We detected the novel c.4269+2_4269+3delTG germline splicing variant in NF1, which proved to be pathogenic by the minigene assay showing an aberrant splicing. The tumor showed a copy-number (CN) neutral loss of heterozygosity for the WT allele, and both ARMM and metastases carried several CN gains associated with NF1-driven carcinogenesis and very low mutation burden. The tGEP analysis unveiled a macrophagic infiltration, with a pro-inflammatory M1-type polarization, in the context of lack of PD-L1 expression.

Conclusions: The response to nivolumab in a germline NF1-driven ARMM case seems independent from levels of TMB and PD-L1 expression and may be mediated by inflammatory response induced by M1-polarized macrophages.

Keywords: M1 macrophages; NF1 syndrome; PD-L1 negativity; anorectal mucosal melanoma; low TMB; nivolumab.

Figure 1.

A) Clinical timeline of the proband. We report the most representative steps of the proband clinical evolution. CT: computed tomography, PR: Partial Response, CR: Complete Response, MRI: Magnetic Resonance Imaging. B) Schematic representation of the hybrid minigene construct used in the experiments. PCR fragments including exon 32 adjacent to NF1 c.4269+2_4269+3delTG variant and at least 350 bp of the upstream and downstream introns were amplified from wild-type genomic DNA and cloned into the β-globin vector, which was obtained by the cloning of the β-globin gene inside a pcDNA™3.1/Hygro(+) vector (ThermoFischer Scientific). Β: β-globin, Ex.: exon, Int.: intron. C) Sanger sequencing of the RT-PCR products obtained after cell transfection with the minigene constructs. HEK293 cells were transfected with the minigene vector harboring the c.4269+2_4269+3delTG variant (MUT) or with the corresponding wild-type one (WT). After RNA extraction and retrotranscription, the resulting cDNA was amplified using β-globin specific primers. On the left, a schematic representation of RT-PCR products obtained, while on the right the corresponding Sanger electropherograms. Junctions between exons are indicated above each sequence.

Figure 2.

A) Chromosome 17 variant allele frequency (VAF) distribution. Paired t-test identified a VAF shift of germline-annotated variants between WB and ARMM#1. B) FACETS output for TSO500 targeted sequencing. Allelic phasing identified a copy number neutral LOH for the NF-1 gene locus. In the copy number (CN) plot, red lines represented the total CN, whereas the black ones the minor allele CN for each genomic segment. C) CUTseq trace for ARMM#1. Shallow whole genome sequencing identified several CN gains across genome, with CNA genes related to a NF-1 driven carcinogenesis annotated in the Fig..

Figure 3.

A) Spider plot of (ss)GSEA significant genesets. TNFA and JAK-STAT3 pathway were enhanced in ARMM, whereas IFN-γ and INF-α signaling only in in all the lesions. The y-axis of the splider reported the NES by ssGSEA. B) Heatmap reporting immune cell-type profiles. Primary and metastatic lesions were highly infiltrated for macrophages and CD8 T-cells. C) ARMM#1 H&E section and IHC staining. Clockwise, we report H&E, CD8, PDL1 and CD68 10X pictures. D) Heatmap of macrophages M1 and M2 related genes. Proinflammatory and M1 markers where enriched in all the lesions.