Frequent alterations in p16/CDKN2A identified by immunohistochemistry and FISH in chordoma

Abstract

The expression of p16/CDKN2A, the second most commonly inactivated tumour suppressor gene in cancer, is lost in the majority of chordomas. However, the mechanism(s) leading to its inactivation and contribution to disease progression have only been partially addressed using small patient cohorts. We studied 384 chordoma samples from 320 patients by immunohistochemistry and found that p16 protein was lost in 53% of chordomas and was heterogeneously expressed in these tumours. To determine if CDKN2A copy number loss could explain the absence of p16 protein expression we performed fluorescence in situ hybridisation (FISH) for CDKN2A on consecutive tissue sections. CDKN2A copy number status was altered in 168 of 274 (61%) of samples and copy number loss was the most frequent alteration acquired during clinical disease progression. CDKN2A homozygous deletion was always associated with p16 protein loss but only accounted for 33% of the p16-negative cases. The remaining immunonegative cases were associated with disomy (27%), monosomy (12%), heterozygous loss (20%) and copy number gain (7%) of CDKN2A, supporting the hypothesis that loss of protein expression might be achieved via epigenetic or post-transcriptional regulatory mechanisms. We identified that mRNA levels were comparable in tumours with and without p16 protein expression, but other events including DNA promoter hypermethylation, copy number neutral loss of heterozygosity and expression of candidate microRNAs previously implicated in the regulation of CDKN2A expression were not identified to explain the protein loss. The data argue that p16 loss in chordoma is commonly caused by a post-transcriptional regulatory mechanism that is yet to be defined.No conflicts of interest were declared.

<p>Deficiency of <em>PTEN</em> and <em>CDKN2A</em> Tumor-Suppressor Genes in Conventional and Chondroid Chordomas: Molecular Characteristics and Clinical Relevance</p>

“…In addition to chromosomal aberrations in the PTEN and CDKN2A loci, some studies evaluated the expression levels of the PTEN and CDKN2A proteins using immunohistochemistry or Western blotting. 15,[34][35][36][37][38] Based on the reported results in these previous studies, PTEN staining was negative in 13%~55% of chordomas, 34,35,37,38 and immunoreactivity for CDKN2A was absent in 53%~96% of chordomas. 15,35,36 However, only two studies analyzed the correlation between the protein expression levels and clinical prognosis.…”

“…15,[34][35][36][37][38] Based on the reported results in these previous studies, PTEN staining was negative in 13%~55% of chordomas, 34,35,37,38 and immunoreactivity for CDKN2A was absent in 53%~96% of chordomas. 15,35,36 However, only two studies analyzed the correlation between the protein expression levels and clinical prognosis. 36,38 Sommer et al found no significant difference in median disease-free survival between CDKN2A-positive chordomas (90 months) and CDKN2Anegative chordomas (24 months).…”

“…12 In the following years, chromosome segments 9p and 10 have become the hotspot regions for studying genetic alterations in chordomas. Many scholars analyzed these chromosomal abnormalities in chordoma tissues and cell lines using various technical platforms, such as aCGH, 6,7,13 FISH, [13][14][15] PCRbased microsatellite loss of heterozygosity (LOH) analysis, 16 GeneChip array, 9 or genome-wide high-resolution SNParray. 17,18 Accumulating evidence has revealed a frequency of PTEN deletion in chordomas ranging from 19% to 80%, and the frequency of CDKN2A deletion in chordomas has been reported to range from 12% to 80%.…”

“…38 Notably, a recent study proposed that the absence of CDKN2A protein expression could not be explained by copy number loss of the CDKN2A gene. 15 In particular, there has been a paucity of studies concerning genetic alterations of PTEN and CDKN2A in distinct histological subtypes of chordoma. Therefore, we designed this study to investigate the molecular characteristics and expression levels of PTEN and CDKN2A in conventional and chondroid chordomas, and the clinical relevance was analyzed.…”

Single-Cell Transcriptome Profiling Reveals Intratumoral Heterogeneity in Human Chordomas

“…We found that clusters 2, 3, and 5 were devoid of SMARCB1 ( Figure S4D). Neither these three clusters expressed CDKN2A, which was frequent lossed in chordomas [38] ( Figure S4E). Next, we conducted a pseudo time analysis of 6 clusters to investigate their developmental trajectories, and we found that clusters 2, 3 and 5 tended to be in a more advanced stage ( Figure 2D, Figure S4F).…”