Clonality analysis of pituitary adenomas: a pilot study
I Baciu1, S Radian1,2, C Capatina1,2, I Botusan1,2, D Aflorei2, L Tataranu3, V Ciubotaru3 & M Coculescu1,2
Introduction: Monoclonality of pituitary adenomas is an established fact. Still, there are exceptions and polyclonality may correlate with aggressive tumor behavior. In order to test association of clonality with pituitary adenoma characteristics in a series from Romania, we implemented a protocol for X-chromosome inactivation analysis at the androgen receptor (AR) locus (HUMARA) and validated it in a number of tumor samples.
Objective: To establish and validate a protocol for pituitary tumor clonality assessment using HUMARA.
Subjects and methods: We tested blood DNA samples from 77 pituitary adenoma female patients and 72 healthy female controls, for assessment of average heterozigosity and identification of AR alleles in our population. DNA was extracted from RNAlater-conserved tissue, for 4 somatotroph tumors (ACM) and 3 nonfunctioning adenomas (NFA). For HUMARA, DNA was incubated with HpaII or water (control) and fluorescent-labeled PCR products of AR exon1 (including the CAG repeat and 2 HpaII sites), were sized with the CEQ8000 Analyzer (Beckman) and converted to CAG-repeat numbers. The clonality ratio was calculated from the areas-under-curve of heterozygous alleles, comparing HpaII-digested versus non-digested DNA. Values <0.4 indicate monoclonality.
Results: For blood DNA, average heterozigosity was 84.7%; median (range) CAG repeats was 23 (1530), in controls (n=72), and 92.21%; 22 (1129) in pituitary adenoma patients (n=77), respectively. HUMARA revealed that 4 out of 7 adenomas were monoclonal, 2 somatotropinomas appeared polyclonal and clonality of 1 sample could not be assessed (HpaII-digested DNA did not amplify).
Conclusions: The HUMARA protocol proved reliable for RNA-later conserved pituitary adenoma tissue and will be used in a larger study. The 2 polyclonal adenomas need confirmation by HUMARA of micro-dissected tissue, in order to exclude contamination with normal cells.
This work was supported by CNCSIS TE 227/2010 and PNCDI2 41-014/2007 grants from the Romanian Ministry of Education, Research and Youth.