Expression of CD271 in genetically unstable patient melanomas is highly variable and not linked to tumorigenic potential in an assay-dependent manner

Some cancers are thought to follow a cancer stem cell (CSC) model in which hierarchical and epigenetically determined relationships exist between phenotypically and functionally distinct cells within tumors. In melanoma, for example, CD271/p75/NGFR (nerve growth factor receptor) was found by some groups to be expressed in cells with enriched tumorigenic potential (Boiko et al., 2010; Civenni et al., 2011). However, these observations were not reproduced in highly efficient patient-derived xenograft (PDX) assays (Quintana et al., 2008), in which plastic, nonhierarchical relationships were proposed between CD271- and CD271+ cells (Quintana et al., 2010). Here we confirm that a CD271-based CSC model does not apply to melanoma, regardless of the PDX assay method used. In side-by-side testing of published PDX assays, no consistent differences were seen in CD271 expression or in PDX tumor formation from purified CD271- or CD271+ cells from patient melanomas. Analysis of CD271 in sixty-eight PDX tumors grown from CD271- or CD271+ cells revealed strikingly variable CD271 re-expression patterns, which were not consistent with stable hierarchical or plastic relationships between CD271-defined cell sub-populations. Genotyping of sibling PDX tumors showed extensive (28% - 48%) genomic copy number differences, which were also apparent (1.4% - 23%) between CD271- and CD271+ cells purified from each of four melanomas, revealing intra-tumoral genetic heterogeneity associated with CD271 expression. Thus, CD271 is not linked reproducibly to tumorigenic potential in melanoma, regardless of the method of melanoma cell isolation, and its expression appears driven by complex and unstable interactions between changing epigenetic and genetic states. Understanding of melanoma biology and the treatment of melanoma patients are unlikely to be advanced by applying a CD271-based CSC model to this disease. To test whether differences in CD271 expression among PDX melanomas grown from CD271- or CD271+ cells could be linked to genetic differences, we genotyped Lin- cells sorted by flow cytometry from four pairs of tumors. Tumors in each pair were grown from aliquots from the same pools of cells, purified according to CD271 expression from patient melanomas 12-1036 and 12-1254. Genomic DNA was extracted from purified Lin- cells from each tumor and subjected to single nucleotide polymorphisms (SNP) genotyping using Illumina Human OmniExpress 715K arrays. To test for genetic differences between CD271- and CD271+ cells within the same tumor, we flow cytometrically-purified CD271- and CD271+ cells from patient (n=1) and PDX (n=4) melanomas, extracted DNA and subjected to SNP genotyping using Illumina 2.58M arrays.