Alpha CTX as a biomarker of skeletal invasion of breast cancer:: Immunolocalization and the load dependency of urinary excretion

We recently showed that increased urinary excretion of the cross-linked, nonisomerized form of the C-telopeptide of collagen type I (alpha alpha CTX) could be a sensitive indicator of the presence of bone metastases in breast cancer patients. The present study was sought to investigate (a) the localization of alpha CTX epitopes in the proximity of a bone metastasis and (b) the relationship between number of metastases and the urinary excretion of alpha alpha CTX. Adjacent bone sections from breast cancer patients were stained for the presence of tumor cells (anti-cytokeratin antibody), osteoclasts (TRAcP activity), and alpha CTX (anti-alpha CTX antibody). The association between the extent of metastatic bone disease and urinary excretion of alpha alpha CTX measured with ELISA was assessed in 90 breast cancer patients (43 with bone metastasis and 45 without bone metastasis). Immunohistochemistry revealed accumulation of TRAcP-pdsitive osteoclasts and intense staining for alpha CTX epitopes in the proximity of cytokeratin-positive bone metastasis. Areas of alpha CTX staining showed unstructured bone tissue under polarized light. In addition, there was a significant linear association between the number of bone metastases and the urinary levels of alpha CTX in breast cancer patients with metastatic bone disease, independent of age and body mass index (r = 0.56, P < 0.001). The estimated relative increases in alpha alpha CTX associated with the presence of one, two, or three metastases are 38%, 57%, and 81%, respectively. Taken into account the 17% intraindividual variation of the assay, alpha alpha CTX could be a sensitive biochemical marker for the close monitoring of cancer patients aiming the facilitation of early metastasis detection.