Plasma TGFβ₁during pelvic radiotherapy for gynaecological malignancy

Abstract

The pro-fibrotic cytokine transforming growth factor b 1 (TGFb 1 ) is implicated in the pathogenesis of radiation fibrosis [1 Á6], which contributes to late morbidity following radiotherapy. Relatively few laboratories have conducted studies during radiotherapy and none to our knowledge during pelvic radiotherapy. Neither have strict precautions been observed to minimise platelet degradation and supra-physiological TGFb 1 release. Excessive platelet degranulation during standard venepuncture causes immediate release of TGFb 1 and further increments can occur during long-term storage of blood/serum unless samples are frozen at (/208C. These factors hinder reliable inter-comparisons of results from different laboratories.Here we report a detailed study involving eight patients: five had cervix cancers and received radical radiotherapy (at 1.8 Gy per fraction) and weekly cisplatinum chemotherapy (numbers 2, 4, 5, 7 and 8), while three received pelvic radiotherapy after hysterectomy for endometrial malignancy (numbered 1, 3 and 6). Following informed consent procedures, venous blood was collected without a tourniquet, through a wide-gauge butterfly needle into a prechilled CTAD tube. Vacutainers and syringes were not used because of the turbulence (and platelet degradation) they induce. Samples were gently mixed by inversion and placed immediately on ice. Within one hour samples were centrifuged for 30 min at 1200 )/g at 4 o C. One millilitre aliquots of supernatant were stored at (/70 o C. All plasma samples were assayed for TGFb 1 within six months of collection in order to minimise the increases during long-term storage which have been reported in previous studies and which are thought to result from release of covalently bound TGFb 1 from a-macroglobulin [7]. In order to avoid inclusion of latent TGFb 1 complexes in human plasma, we used the R&D Quantikine ELISA System for human TGFb 1 , with pre-treatment of samples with 2.5N acetic acid/10M urea to disrupt a range of TGFb 1 complexes. Each sample was assayed in duplicate at three serial dilutions and the mean and standard error calculated from the resulting six readings.The baseline plasma TGFb 1 results (see Figure 1) were generally lower than the published data relating to lung and breast cancer, which is consistent with our sample collection technique. The standard error values are small. Most remained below 5 ng/ml during treatment, but two patients had levels /10 ng/ml. Close inspection of Figure 1 reveals that there are possible cyclical fluctuations in plasma TGFb 1 . Such fluctuations could be due to random variation (or are associated with the assay technique), rather than being a result of ordered homeostatic cyclical activity. Another possibility is that results were heavily influenced by the weekly sampling times: for example, if twice weekly sampling had been used, then further fluctuations and a shorter cyclical period might have been found. One patient (number 5) with recurrent cancer and who had undergone previous radiotherapy and extensive pelv...