Elmar Lindhorst

Molecular detection of thyroglobulin mRNA transcripts in peripheral blood of patients with thyroid disease by RT-PCR

The sensitive detection of circulating tumour cells in patients with differentiated thyroid cancer may precede the detection of relapse by other diagnostic studies – such as serum thyroglobulin – and thus may have important therapeutic and prognostic implications. We performed reverse transcription-polymerase chain reaction (RT-PCR) on blood samples from patients diagnosed with thyroid disease using two different RT-PCR sensitivities. Additionally, tissue specificity of TG mRNA-expression was determined using RNA extracts from 27 different human tissues. The lower limit of detection was 50–100 TG mRNA producing cells/ml blood using a ‘normal’ RT-PCR sensitivity and 10–20 cells/ml blood using a ‘high’ sensitivity. With the normal sensitivity TG mRNA was detected in 9/13 patients with thyroid cancer and metastasis, 63/137 patients with a history of thyroid cancer and no metastasis, 21/85 with non-malignant thyroid disease and 9/50 controls. With the high sensitivity TG mRNA was detected in 11/13 patients with thyroid cancer and metastasis, 111/137 patients with a history of thyroid cancer and no metastasis, 61/85 with non-malignant thyroid disease and 41/50 controls. Interestingly, using the normal RT-PCR sensitivity TG mRNA transcripts are specific for thyroid tissue and detectable in the peripheral blood of controls and patients with thyroid disease, which correlates with a diagnosis of metastasized thyroid cancer. However, with a high RT-PCR sensitivity, TG mRNA expression was found not to be specific for thyroid tissue and was not correlated with a diagnosis of thyroid cancer in patients. As a consequence, to date TG mRNA detected by RT-PCR in the peripheral blood cannot be recommended as a tumour marker superior to TG serum-level.

115 CARTILAGE BIOMARKERS AFTER MENISCECTOMY

Purpose: Study of 846 as anabolic marker and C2C as catabolic marker of post-traumatic osteoarthritis (OA) in the rabbit model of anterior cruciate ligament transection (ACLT). Methods: Transection of the anterior cruciate ligament was performed in adult male New Zealand white rabbits (n = 32). An open arthrotomy surgery of only the right knees was used. Left knees remained untreated and served as intraindividual control knees. At time 0 before surgery and at 2, 4, 8 and 12 weeks before sacrifice, synovial fluid was aspirated from both, right and left knee joints. Use of all animals had been permitted by both, the University’s ethics committee and the government ethics committee (Regierungsprasidium Darmstadt, Germany). Macroscopic grading was performed of all right and left knee joints. 9 areas per each medial and lateral tibia, respectively medial and lateral femur and the patella were evaluated. Histologic grading was performed with a grading system accounting for proteoglycan content, matrix structure, cellularity, and osteophyte formation. 846 levels and C2C levels were measured in the synovial fluid lavages with immunoassays. The distributions of synovial fluid levels of 846 and C2C were statistically analysed (Kolmogorov-Smirnov-test). Differences between left and right knees were then statistically analysed by student’s t-test. Results: All rabbits had a normal postoperative course after ACLT. At 2 weeks after ACLT, macroscopically visible lesions were only detected at the medial femoral site (p< 0.05). With time, lesion size increased and lesions appeared at more joint sites. At 12 weeks after ACLT, medial and lateral tibiae as well as both femoral condyles showed statistically significant lesions. Histologic scores showed early OA after ACLT, when compared with the unoperated contralateral side. 846 levels in the synovial fluid lavages of the operated knee joints were increased at all timepoints (p< 0.05) when compared to the non-operated contralateral knees. The highest levels were measured from 2 to 8 weeks with a decrease at 12 weeks. The C2C levels at 4 and 8 weeks were higher than at 2 weeks after ACLT (p< 0.05). Conclusions: To our knowledge, this is the first report of 846 and C2C studied in the same rabbit synovial fluid samples. Both markers have been reported as potential markers of metabolic processes in post-traumatic OA, probably reflecting degrading, respectively rebuilding cartilage processes. Biologically, they might be markers of the ensuing macroscopic and microscopic changes. While the reparative response, as possibly indicated by 846, is not sufficient to stop the destructive process of OA, possibly indicated by C2C, the exact meaning of these marker levels has to be characterised in more detail.

114 CARTILAGE BIOMARKERS AFTER ACLT

Purpose: Study of 846 as anabolic marker and C2C as catabolic marker of post-traumatic osteoarthritis (OA) in the rabbit model of anterior cruciate ligament transection (ACLT). Methods: Transection of the anterior cruciate ligament was performed in adult male New Zealand white rabbits (n = 32). An open arthrotomy surgery of only the right knees was used. Left knees remained untreated and served as intraindividual control knees. At time 0 before surgery and at 2, 4, 8 and 12 weeks before sacrifice, synovial fluid was aspirated from both, right and left knee joints. Use of all animals had been permitted by both, the University’s ethics committee and the government ethics committee (Regierungsprasidium Darmstadt, Germany). Macroscopic grading was performed of all right and left knee joints. 9 areas per each medial and lateral tibia, respectively medial and lateral femur and the patella were evaluated. Histologic grading was performed with a grading system accounting for proteoglycan content, matrix structure, cellularity, and osteophyte formation. 846 levels and C2C levels were measured in the synovial fluid lavages with immunoassays. The distributions of synovial fluid levels of 846 and C2C were statistically analysed (Kolmogorov-Smirnov-test). Differences between left and right knees were then statistically analysed by student’s t-test. Results: All rabbits had a normal postoperative course after ACLT. At 2 weeks after ACLT, macroscopically visible lesions were only detected at the medial femoral site (p< 0.05). With time, lesion size increased and lesions appeared at more joint sites. At 12 weeks after ACLT, medial and lateral tibiae as well as both femoral condyles showed statistically significant lesions. Histologic scores showed early OA after ACLT, when compared with the unoperated contralateral side. 846 levels in the synovial fluid lavages of the operated knee joints were increased at all timepoints (p< 0.05) when compared to the non-operated contralateral knees. The highest levels were measured from 2 to 8 weeks with a decrease at 12 weeks. The C2C levels at 4 and 8 weeks were higher than at 2 weeks after ACLT (p< 0.05). Conclusions: To our knowledge, this is the first report of 846 and C2C studied in the same rabbit synovial fluid samples. Both markers have been reported as potential markers of metabolic processes in post-traumatic OA, probably reflecting degrading, respectively rebuilding cartilage processes. Biologically, they might be markers of the ensuing macroscopic and microscopic changes. While the reparative response, as possibly indicated by 846, is not sufficient to stop the destructive process of OA, possibly indicated by C2C, the exact meaning of these marker levels has to be characterised in more detail.