KRAS, EGFR, PDGFR-α, KIT and COX-2 status in carcinoma showing thymus-like elements (CASTLE)

Tissue microarrays (TMA) were performed from the paraffin-wax-embedded blocks of the tumor specimens. A tissue arrayer device (Beecher Instruments, Sun Prairie, WI, USA) was used. All the investigated cases were reviewed and representative tumor areas were marked in the corresponding paraffin wax blocks, of which at least two were sampled. The diameter of the cylinders was 1.2 mm. For immunohistochemistry 5 μm sections on poly-L-lysine-coated slides were used after drying in an oven, followed by dewaxing and peroxidase blocking with 1% H₂O₂. The specificity of immunostaining was tested by replacing primary antisera with normal rabbit serum. No additional pre-treatment procedure for antigen retrieval was done. The immunoreactions were performed with the following primary monoclonal antibodies: Anti-COX-2 (monoclonal rabbit IgG; Santa Cruz, USA; 1:50), anti-CD5 (monoclonal mouse IgG; Novocastra, Leica Microsystems, Wetzlar, Germany; 1:25), anti-TTF-1 (monoclonal mouse, IgG, SPT24; Menarini, Florence, Italy; 1:100), anti-CK 5/6 (monoclonal mouse IgG, M7237; Dako Cytomation, Glostrup, Denmark; 1:50), anti-Ki67 (monoclonal mouse IgG, K-2, Zytomed, Berlin, Germany; ready-to-use). Primary polyclonal antibodies used were: CD117 (polyclonal rabbit, A4502; Dako Cytomation, Glostrup, Denmark; 1:50), anti-thyroglobulin (polyclonal rabbit, A0251; Dako Cytomation, Glostrup, Denmark; 1:80000) and anti-calcitonin (polyclonal rabbit, A0576; Dako Cytomation, Glostrup, Denmark; 1:4000). After washing steps signal detection was performed (EnVision + system-HRP, AEC, Dako Hamburg, Germany; 1:200) followed by counterstaining in hemalaun. Detection of EGFR was performed with the EGFR pharmDX kit (Dako Hamburg, Germany) according to manufacturer’s instructions.

The presence of clearly visible dark brown precipitation was considered as immunoreaction, evaluated by a score for staining intensity (0: no staining; 1: weak; 2: moderate; 3: strong reaction intensity).

Cores of tumor tissue selected from regions on correspondent Hematoxylin-Eosin slides were manually punched out from the donor blocks. DNA was extracted from paraffin-embedded tissue sections using EZ1 DNA tissue Kit (Qiagen, Hilden Germany) after pre-treatment with proteinase K (Qiagen, Hilden Germany). 150 ng DNA were used for PCR amplification and all PCR reactions were run at a final volume of 25 μl (Red Jump MasterMix, Sigma-Aldrich). Exons 9, 11, 13 and 17 of KIT, exons 10, 12, 14 und 18 of PDGFR-α, exons 18, 19, 20 and 21 of EGFR and exons 2 and 3 of KRAS were evaluated for the presence of mutations by PCR amplification and direct sequencing. The primer pairs used for PCR amplification and direct sequencing are shown in Table 1. After estimation of DNA concentration by spectroscopy (GeneQuant) relevant exons were amplified by PCR under standard conditions with an annealing temperature of 50°C for Exon 17 (KIT), 55°C for Exon 2 and 3 (KRAS), Exon 9 (KIT) and Exon 18 (PDGFR), 60°C for Exon 18, 19, 20 and 21 (EGFR) and Exon 11 and 13 (KIT) as well as Exon 10, 12 and 14 (PDGFR) with the corresponding primers. The PCR products were then separated on agarose gel containing ethidiumbromide and visualized under ultraviolet light. Afterward PCR products were purified using high pure PCR product purification kit (Roche Diagnostics, Penzberg, Germany) and then sent to Qiagen (Hilden, Germany) for sequencing according to Sanger method.

This study was approved by the ethics committee of the University Hospital Heidelberg (reference number: 206/2005).

The primary symptom was a palpable node in 2 patients and an adenomatous goitre as well as a paresis of the recurrent nerve in 1 case, respectively. In 3 cases no case history was available. 5 tumours were located in the thyroid and 1 was partially extrathyroidal, the latter with the majority of the tumor mass in the thyroid gland.

Local tumor stage was T2 in one case, T3 in 3 cases, the remaining cases T4. In 3 cases no lymph nodes were affected, 3 patients already developed lymph node metastases. All cases underwent curative hemi- or total thyroidectomy. 5 patients received postoperative radiotherapy, 1 received a combined radio- and chemotherapy. The median time of follow-up observation was 18.5 months, with a range from 2 to 36 months. During this time, 1 patient developed local recurrence 2 months after primary operation and underwent a second tumorectomy with bilateral neck dissection and transsternal lymphadenectomy. The remaining patients were recurrence-free (see Table 2). By histology the resected specimen showed fibrous septa with lobulation and surrounding tumor islands of various size (see Figure 1C + F). Especially the peripheral tumor tissue was infiltrated by small lymphocytes (see Figure 1A,B,D,E). Scattered blood vessels were found, surrounded by fibrous connective tissue.Tumor cells were varying in shape: Irregular cells as well as spindle-shaped and epithelioid cells were observed. Occasionally, squamoid or spindle-shaped epithelial cells with whorls resembling Hassall’s corpuscles were noticed. The cytoplasm of the tumor cells was pale and nuclei were clearly evident and slightly oval (see Figure 1A - F).

All CASTLE cases expressed CD5 in a moderate to strong reaction and CD117 in predominantly weak to moderate intensity in the epithelial component. Only in one case a strong staining signal for CD117 could be detected. (see Figure 1G + H). Staining with CK5/6 showed weak to strong reactions in all samples (see Figure 1I). EGFR showed a strong reactivity in three, weak staining intensity in two samples and no staining pattern in one case (see Figure 1J). COX-2 was detected positively in all cases with mainly moderate expression patterns (see Figure 1 K). The expression of Ki67 showed varying results between 2% and 85%. The staining results for CD117, EGFR, COX-2 and Ki67 are shown summarized in Table 3. All cases were negative for calcitonin, thyroglobulin and TTF-1 (Figure 1L).

Mutational analysis of KIT, PDGFR-α, EGFR and KRAS was possible in only 4 cases due to inapplicable DNA in case number 2 and 3.

Mutation analysis of all four investigated cases revealed the absence of an activating mutation in the PDGFR-α gene of exon 10, 12, 14 and 18. Nevertheless, in all four cases a silent single nucleotide polymorphism with a base substitution (CCA > CCG) in exon 12 at codon 567 (p.P567P) of the PDGFR-α gene was observed. This change has already been described in the SNP database with the number rs1873778 (Figure 2A). In 3 (3/4) patients a silent single nucleotide polymorphism with a base substitution (CAG > CAA) in the EGFR gene of exon 20 at position 787 (p.Q787Q) was observed, which has also already been described in the SNP database with the number rs1050171 (Figure 2B). No mutation associated with drug sensitivity in the hotspot exons 18, 19, 20 and 21 was detected. No patient had detectable sequence variants for KIT in the hotspot region of exon 9, 11, 13 and 17. Also no sequence variants were found in the sequenced exons of the KRAS gene (Table 4).