Italiano et al. BMC Cancer 2012, 12:408 http://www.biomedcentral.com/1471-2407/12/408 Protocols and primers are available on request. Sequence analysis was performed with Applied Biosystems Se- quence ScannerTM v1.0. Western blotting Western blotting was performed to assess the expression of SDHA in WT GIST with and without SDHA muta- tion. Frozen tissue from seven WT GIST (two with SDHA mutation and five without SDHA mutation) were homogenized in RIPA buffer supplemented with prote- ase and phosphatase inhibitors. Electrophoresis and im- munoblotting were performed on the protein extracts using 30 μg of protein per sample and the anti-SDHA rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was diluted according to the manu- facturers’ recommendations. Following hybridization with the secondary anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the blots were incubated with Immun-Star horseradish peroxidase luminal/enhancer (Bio-Rad, Hercules, CA, USA) and exposed onto Kodak Biomax MR Film (Eastman Kodak Co., Rochester, NY, USA). Immunohistochemistry Immunohistochemistry (IHC) was applied on all 17 WT GIST cases tested using SDHA (Abcam, Cambridge, UK, 1:1000) and SDHB (Sigma-Aldrich, St Louis, MO, 1:500), according to manufacturer’s recommendations. The results of the SDHA and SDHB immunohistochem- istry were recorded blindly to the KIT, PDGFRA or SDHA genotyping. One KIT exon 11 mutant GIST from a young adult patient was used as control. The immu- noreactivity was scored as negative (loss of expression) if the tumor cells were negative but the entrapped normal tissues (endothelial cells) stained positive. Conversely, a positive result (retained expression) was interpreted if the tumor cells showed the same intensity staining as the internal positive control cells. If the tumor showed a weak intensity of staining, significantly lower than the normal tissue, the result was interpreted as partial loss of expression. Results Mutation of SDHA is a recurrent event in young adults with KIT and PDGFRA WT GIST Massive parallel next-generation sequencing of six cases of WT GIST (five pediatric and one young adult) revealed that the GIST from the young adult patient (22 year-old man, with multinodular gastric lesions and multiple liver metastases) carried a C to T transition at nucleotide 206 in SDHA exon 2, a nonsense mutation resulting in the replacement of ar- ginine with a stop codon at residue 31 of SDHA, causing truncation of the peptide chain at residue 30 Page 3 of 7 (p.Arg31X) (Figure 1). This result was then validated by targeted SDHA exon 2 Sanger sequencing from the DNA isolated from both tumor and normal tis- sue, in keeping with a germline mutation. Of note, the SDHA sequence electropherogram of the normal DNA revealed equivalent proportion of the wild-type and the mutated allele (T), whereas tumor DNA con- tained predominantly the mutated allele (T), indicat- ing relative loss of the wild-type SDHA allele. This patient is alive with disease 66 months after the ini- tial diagnosis, and was treated with multiple kinase inhibitors with marginal responses, including imatinib, sunitinib, sorafenib and sirolimus, and being presently on regorafenib. Since two prior reports also identified mutations in SDHA exons 9 and 13 [16,17] in WT GIST, we per- formed targeted SDHA exons 2, 9 and 13 sequencing in 11 additional cases of pediatric and young adult WT GIST. By this method, another young adult WT GIST (26 year-old woman with bulky intra-peritoneal and liver metastatic disease) was identified to harbor a missense mutation in SDHA exon 2 (p. D38V) (Figure 2). In this latter case, the mutation was observed only in the tumor and not in the normal tissue DNA tested. This patient was alive with disease 15 years after the initial diagnosis, preceding the availability for targeted therapy. No additional muta- tions in SDHA exons 9 or 13 were identified. Mutation of SDHA in WT GIST is associated with concomitant loss of both SDHA and SDHB proteins expression To confirm the functional impact of SDHA mutation, we assessed SDHA protein expression by western blotting in seven WT GISTs, including three samples from the two patients with SDHA mutation and four without SDHA mutation (Figure 3). We found that SDHA expression was absent in the two cases har- boring a mutation of SDHA and present in the other cases. In addition, immunohistochemistry for SDHA and SDHB was performed. The WT GIST associated with a germline SDHA mutation showed complete loss of both SDHA and SDHB protein (Figure 4 A,B), while the tumor with a somatic, heterozygous SDHA mutation showed significant decreased in SDHA immuno-expression, as well as complete loss of SDHB (Figure 4 C,D). In contrast, strong and diffuse SDHA reactivity was present in all WT pediatric and young adult GIST tumors tested without detectable SDHA mutations, which matched with a complete loss of SDHB expression (Figure 4 E,F). Furthermore, both SDHA and SDHB expression was preserved in a control case of a young adult GIST carrying a KIT exon 11 deletion (Figure 4 G,H).