ANÁLISIS DE RIESGOS EN EL CASO DE ESTUDIO

joints) as measured in degrees on the Dupuytren affected hands (n=21) is depicted on

the y-axis.

(O 0) 2 S' ■o § % "D C m o 1 6 0 140 120 100 80 60 40 20 0

c-myc vs total joint angle deformity in recurrent disease

♦ ♦

0 10 20 30 40 50 60 70 80 90

c-myc

Figure 7:C-myc expression in recurrent Dupuytren's disease related to severity.

Ihe x-axis represents the c-myc activity as a percentage as described in figure 5 whilst total joint deformity (metacarpophalangeal, proximal and distal interphalangeal joints) as measured in degrees on the Dupuytren affected hands (n^9) is depicted on

the y-axis.

C-myc levels for Dupuytren's disease were not significant using the paired t-test if plotted against patients age or number o f operations in recurrent disease.

5.

Discussion

It has been suggested that cellular c-myc expression is dependant on the presence o f mitogens (reviewed by Evan and Littlewood 1993), except in transformed cells which do not require mitogenic stimulation for c-myc expression (Campisi et al 1984). C-myc is also though to be expressed at a constant rate throughout the cell-cycle in proliferating cells (Hann et al 1985, Thompson et al 1985), but not in quiescent fibroblasts (Campisi et al 1984, Dean et al 1986, Waters et al 1991). C-myc is thought to mediate control o f cell-cycle progression, proliferation and apoptosis (Reed 1994) and once these processes were established, Evan and Littlewood (1993) suggested that the cell-growth and cell- death could be speculated to be independently modulated by other genes, cytokines and other external factors.

Levels o f c-myc expression could therefore hypothetically be interpreted as a measure of cellular activity (high cell turn-over) and Fibrosarcoma specimens would accordingly be expected to exhibit very high levels of c-myc which were confirmed. By contrast carpal ligament representing tissue with a low level of cellular turnover, exhibited very low levels o f c-myc. Primary Dupuytren’s disease tissue exhibited a high level o f c-myc expression not significantly different from Fibrosarcoma specimens and would therefore be expected to possess a similar degree of cellular turnover as the malignant tumour . According to the theories discussed above, the high c-myc expression would indicate that Dupuytren fibroblasts were either under the influence o f mitogens, represented transformed cells or proliferating.

Recurrent Dupuytren's disease and non-diseased fascia showed intermediate levels o f c- myc which could indicate a lower availability of or lower sensitivity to mitogens,

represented by a higher ratio of quiescent to proliferating cells. Considering the disease progresses fi'om non-diseased fascia to primary Dupuytren's disease and then reappears as recurrent Dupuytren's disease, it could be hypothesised that these intermediate levels could be interpreted in two ways.

If expression of c-myc is dependant on the availability of mitogens, the intermediate level o f c-myc in non-diseased fascia may rise with increased availability o f cytokines. If this is the case therefore, non-diseased fascia would exhibit c-myc expression

mitogens. This may in turn lead to disease progression from non-diseased fascia to primary Dupuytren’s disease.

In contrast it could be hypothesised that the intermediate level o f c-myc expression in recurrent Dupuytren's disease is due to the reduced sensitivity to cytokines. Recurrent Dupuytren’s disease may represent a different tissue, such as scar tissue, which due to its fibrotic nature may be less sensitive to available cytokines.

If the theory of disease progressing from non-diseased fascia to primary Dupuytren’s disease and then reappearing as recurrent Dupuytren’s disease is accepted, then c-myc expression has been shown to fluctuate at different times o f the disease process. Individual measurements o f c-myc expression from nodules and cords were not performed and may therefore account for some difference in these results.

In recurrent Dupuytren’s disease correlation between total joint angle deformity and c-myc levels was highly significant, i.e. the more severe the disease the higher the c-myc expression. The level of contraction encountered may however depend on patient neglect, at which stage the patient seeks medical attention and whether the disease process is recognised as Dupuytren’s disease, though the latter was excluded in this study. Joint contracture remains the basis of Tubiana’s (1974) classification of

Dupuytren’s disease and forms part of the clinical decision to operate. The correlation between total angle deformity and level o f c-myc is however consistent with c-myc expression representing disease activity in Dupuytren’s disease, as measured by joint contraction.

C-myc levels were not found to be correlated to total joint deformity in primary disease and may indicate that alternative criteria for disease severity are applicable in primary disease.

Because c-myc levels were not comparable with the number o f recurrences in recurrent Dupuytren’s disease, it will only become clear whether c-myc expression in primary disease can be used as a predictor of recurrence after long-term follow-up o f primary cases.

The immunochemical methods employed in this chapter are well established and

practiced routinely at the Gray Laboratory at Mount Vernon Hospital, though this does not exclude possible artifacts. Each sample posed as the corresponding internal control in the absence of the myc antibody, but was incubated with rabbit immunoglobulin fraction (Sigma Ltd, UK), in order to act as a baseline for subsequent flow cytometry analysis.

The large amount of collagen in the Dupuytren, non-diseased fascia and carpal hgament samples resulted in an intermittent slow flow through the FACscan, which required frequent flushing of the machine and caused a substantial amount of fluorescent debris. The use o f CGWs however allowed the definition o f specific populations of nuclei and regions were set around populations to omit extraneous interference from debris. These regions were retained and superimposed on the control sample, which allowed

comparison o f the number of nuclei and fluorescence in identical regions.

C-myc levels have been shown to be o f comparable magnitude in Fibrosarcoma and primary Dupuytren specimens, with decreasing levels in recurrent Dupuytren's disease, non-diseased fascia and carpal hgament. C-myc expression in Dupuytren's disease may therefore be taken to fluctuate throughout the disease process, either due to reduced availability o f or reduced sensitivity to cytokines. It is also proposed that primary Dupuytren's disease may represent transformed fibroblasts, which do not require mitogenic stimulation for c-myc expression.

To establish the value o f c-myc as a prognostic marker o f recurrence in Dupuytren's disease long-term follow-up o f primary Dupuytren's disease patients would be necessary.

Measuring c-myc levels on recurrent Dupuytren's contracture was however shown to predict the severity o f the recurrent disease and may be used to facilitate the decision to

Chapter III

Markers of proliferation and anti-apoptotic genes