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Over the past 10 years, several laboratories have examined whether the in-vitro radiosensitivity of both tumour and normal cells is an independent prognostic factor for the outcome of radiotherapy.

1.6.3.1 Tumour studies

Several studies carried out on primary tumours have attempted to evaluate the predictive value of radiosensitivity measurements for radiotherapy outcome. The ATCCS assay (§1.8.5.1.2) has been used by two groups. The first considered patients who underwent post-operative radiotherapy, and SF2 was found not to be predictive of treatment

outcome (Brock et a l 1990). Twelve patients that had local failure after a one year follow-up period had a mean SF2 of 0.40, compared with a mean SF2 of 0.30 in the

controls (60/72) with the difference being statistically insignificant. However surgery may have been a confounding factor in this study. In a second study in which the majority of head and neck patients were treated with radiotherapy alone, tumour radiosensitivity as measured by a was a significant prognostic factor when a high (above median) value was used to stratify the data (Grinsky et a l 1993, 1994). However, an updated report from this group studied 92 patients with a median follow- up of 6 8 months and found no relationship (Eschwege et a l 1997). Two further studies

have used the modified Courtney-Mills soft agar assay. Measurement of tumour SF2 in

128 cervical cancer patients treated with radiotherapy alone with a minimum follow-up time of two years was shown to be an independent prognostic factor for treatment outcome and a significant prognostic factor for the probability of developing late complications (West et a l 1993, 1997). Conversely, a recent study by Stausbol-Gron and Overgaard (1999) examined 38 patients with squamous cell carcinoma of the head and neck and found that disease stage, but not tumour cell SF2 predicted loco-regional

TCP.

1.6.3.2 Norm al tissue

The importance of normal-tissue radiosensitivity and its use in the development of a predictive assay depends upon the hypothesis that a relationship exists between in-vitro cellular and in-vivo normal-tissue radiosensitivity.

Considering extreme over-reactors such as AT homozygotes, Taylor et a l (1975) showed that the in-vitro radiosensitivity of skin fibroblasts from patients with AT was significantly higher than that of normal human fibroblasts. Other early studies reported individuals or small groups of patients who sustained worse than expected radiotherapy reactions (Table 4.7). Weichselbaum et a l (1976) compared the radiosensitivity of fibroblasts established from individuals showing severe reactions to radiotherapy with that of similar cells taken from normal controls and concluded that intrinsic radiosensitivity was not the underlying cause of the observed sensitivity. However, reports from severe over-reactors and HRS patients who were found to have cells more sensitive on average than those from putatively normal donors, led to the conclusion that enhanced radiation sensitivity in vitro correlated with enhanced radiation sensitivity chnically (Woods et a l 1988, Plowman et a l 1990, Brown et a l 1996, Smith et a l

1980, LoefUer et a l 1990). In the study by Little and Nove (1990) three of the five strains derived from patients with a clinical response that was unusually sensitive to X-ray therapy in the Loeffler study fell within the lower range of 114 normals.

Although these early studies represented valid support for the argument that variations in individual radiosensitivity exist and were linked with normal-tissue response, the studies were on small numbers of patients and did not allow a simple evaluation of the predictive capabilities of the in-vitro assay due to the strong patient selection.

A number of studies (reviewed in Budach et a l 1998) have now evaluated the relationship between the range of acute and late normal-tissue reactions observed in vivo and the measured in-vitro normal-tissue radiosensitivity (Table 1.4).

Reference Clinical Endpoint No. of patients (site) Radiation protocol

Cell type and assay endpoint

Correlation with: Correlation Significance acute late Burnet et al. (1992, 1994b) Erythema Telangiecasia 6 (breast) Exponential HDR, LDR Fibroblast ^0 .0 1 Clonogenic yes (coincidental?) yes p<0 . 0 2 Geara et al. (1993)

Late tissue reaction (RTOG)

Acute reactions in skin and mucosa (RTOG)

26 (head and neck, breast) Plateau HDR-IP Fibroblast (SF^ Clonogenic no yes p<0 . 0 0 0 1 Begg et al. (1993)

Erythema 24 (breast) Exponential

HDR Fibroblast (SF2, Do,) Clonogenic no p<0.13 (SF2) p<0 . 1 2 (Dq i) Johansen et al. (1994a)

Subcutaneous fibrosis 1 2 (breast) Exponential

HDR Fibroblast (SF,.,) Clonogenic yes p<0.014 Brock et al. (1995) Erythema Telangiectasia 2 2 (breast) Plateau HDR-IP Fibroblast (SF^) Clonogenic no yes pcO.034"

Table 1.4. Correlation of in-vitro normal-tissue cellular radiosensitivity with acute and late tissue response in vivo. ^average SF2 from duplicate biopsies.

Reference Clinical Endpoint No. of patients (site) Radiation protocol

Cell type and assay

endpoint

Correlation with: Correlation Significance acute late Ramsay and Birrell (1995) Subcutaneous fibrosis (RTOG) Telangiectasia 56 (breast) HDR Lymphocyte (SF^ MTT ■ yes p<0 . 0 2 Johansen et al. (1996) Erythema Subcutaneous fibrosis Telangiectasia 31 (breast) Exponential HDR Fibroblast SF3 .5 Clonogenic no yes'’ p<0.0009 Rudat et al. (1997)

Acute mucosa and skin reactions (WHO classification)

25 (head and neck) Exponential

HDR Fibroblast SF, Clonogenic no Russell et al. (1998)

Subcutaneous fibrosis 79 (breast) Plateau

HDR Fibroblast SF2 Clonogenic Non­ significant trend p<0.13 Peacock (1998)

Late tissue reactions 104 (breast) Plateau

HDR, LDR Fibroblast ^ 0 .0 1 Clonogenic no p<0.19 (LDR)

Table 1.4 continued. Correlation of in-vitro normal-tissue cellular radiosensitivity with acute and late tissue response in vivo. '’correlation with fibrosis, not telangiectasia.

1.6.3.2.1 Acute clinical reactions

Burnet et al. (1992, 1994b) studied fibroblast radiosensitivity in 6 breast cancer patients

selected for large known differences in late radiation reaction from a larger group of patients from the Gothenburg fractionation trial (Turesson 1990). Although the degree of acute reactions correlated significantly with fibroblast radiosensitivity for left sided but not right sided parasternal portals, in a combined data analysis for both sides, no significant correlation was found. Brock et a l (1995) based their studies on a larger sample (n=20) from the Gothenburg series and were unable to detect a relationship between acute skin reaction and fibroblast radiosensitivity whether they used SF2 from

single biopsies or the average values of two biopsies. Begg et a l (1993) studied 24 unselected breast cancer patients but found no correlation between fibroblast sensitivity (SF2 and Do.i) and skin erythema during chest-wall irradiation. Two further studies

examined head and neck cancer patients. Neither Rudat et a l (1997) or Geara et a l (1993) found a correlation between fibroblast sensitivity and in the latter study T-lymphocyte radiosensitivity (measured with the hmiting dilution assay) and acute mucosal and skin reactions.

1.6.3.2.2 Late clinical reactions

Several studies have been published examining the relationship between normal-tissue radiosensitivity in vitro and late radiation reactions in vivo (Table 1.4). Two studies were based on the Gothenburg breast cancer series. Late effects, in particular the development of telangiectasia after chest wall irradiation, have been carefully documented in these patients with a minimal follow-up in excess of ten years. Burnet e t a l (1992, 1994b) studied six of these patients with a range of tissue reactions. Fibroblast low dose-rate (LDR) radiosensitivity was significantly correlated with maximum telangiectasia score at ten years post radiotherapy whether the left or right side parasternal portals or an average of both sides was considered. Brock et a l (1995) measured fibroblast radiosensitivity at both HDR and LDR irradiation and following immediate and delayed plating in 12 patients from the Gothenburg series. Although a negative trend was seen between telangiectasia and fibroblast sensitivity with all assay conditions, it only reached significance with the HDR and immediate plating protocol.

Johansen et a l (1994a, 1996) established fibroblast cultures from a group of 31 unselected breast cancer patients at least ten years after postmastectomy radiotherapy. Development of both fibrosis and telangiectasia was well-documented during a follow-up time of at least five years. A significant negative correlation was found between SF3 5 (which reflected the clinical dose per fraction that these patients received)

Geara et a l (1993) examined the relationship between fibroblast radiation sensitivity and the maximum grade of late reaction in skin, mucosa, soft tissue, bone and larynx in a group of 20 head and neck cancer patients who had received at least 50 Gy. They reported a significant negative correlation irrespective of whether 3 patients who had been selected for the study because of unusually high or low radiation reactions were included. T-lymphocyte radiation sensitivity was also assessed in the above study, but no correlation with late skin, mucosa or soft tissue radiation reactions was found.

Ramsay and Birrell (1995) determined the in-vitro radiosensitivity of transformed lymphocytes (lymphoblastoid cell lines, LCL) from 56 breast cancer patients using the MTT assay (§1.8.5.1.2). Skin and subcutaneous tissue damage was graded from 0-4, where 0 indicated no reaction and 4 maximum reaction. Eleven patients showed late tissue damage (grade 2 ^ ) and the LCL from these patients were more radiosensitive in vitro (SF2) than the other cell lines (p<0.0 2).

Although the above studies suggest that a correlation exists between the cellular radiosensitivity of fibroblasts in vitro and late, but not acute normal-tissue response in vivo, these studies have involved relatively small numbers of patients and in some studies a weak correlation between the in vitro and chnical data has been observed. A further two studies involving larger numbers of patients were carried out to provide definitive information on the nature of the relationship.

Russell et a l (1998) studied fibroblast radiation sensitivity in 79 breast cancer patients selected from a larger group of 385 patients who had undergone breast conservation therapy. Although they found a trend between fibroblast SF2 and the degree of late

fibrosis, this did not reach statistical significance (p=0.13). The second study from the Royal Marsden Tmst examined 104 breast cancer patients, 39 of whom showed significant late reactions. No significant relationship was found between the measured fibroblast radiosensitivity (Dq.oi) and late normal-tissue reactions observed (Peacock

1998).