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Prostaglandins and prognosis in human breast cancer.

Prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) were measured by gas liquid chromatography--mass spectrometry (glc-ms) in extracts of primary tumours from 78 patients with early breast cancer. These levels have been related to factors of established prognostic value and the patients disease-free interval. Although there was a wide variation in amounts of both prostaglandins extracted from different tumours, no significant relationship was observed between levels of prostaglandins and oestrogen receptors (ER), tumour size, presence of lymph node involvement and disease-free interval following primary treatment. It therefore seems unlikely that the level of these particular prostaglandins within breast carcinomas plays a fundamental role in the prognosis of the disease.

Previous studies have shown that human mammary cancers produce greater amounts of 'prostaglandin-like material' than the normal tissues in which they arise (Bennett et al., 1977;Bennett, 1982). It has been suggested that increased prostaglandin levels might be related to tumour growth and to the dissemination of the disease (Bennett et al., 1977;Rolland et al., 1980). However, investigations into the possible prognostic significance of prostaglandins in breast cancers are not consistent. It has been reported that tumour prostaglandin levels are elevated in patients with a poor prognosis (Bennett et al., 1977). Conversely, it has also been suggested that increased prostaglandin levels are associated with favourable prognostic indices (Karmali et al., 1983;Fulton et al., 1982;and Campbell et al., 1982). A further study (Wilson et al., 1980) found no relationship between prostaglandin levels and established prognostic factors.
The reason for such disparity is not completely clear but differences in the methodology used to quantitate prostaglandins may be one important factor. Most investigations have employed bioassay or radioimmunoassay which do not necessarily identify individual prostaglandins. The aim of the present study, therefore, was to measure PGE2 and PGF2a levels in primary breast cancer using the more definitive technique of glc-ms and to correlate these prostaglandin levels with factors of established prognostic value.

Materials and methods
Tumour was obtained from 78 women with early carcinoma of the breast i.e. with no evidence of metastatic disease at the time of presentation. These patients comprised 12 premenopausal, 9 perimenopausal (within 5 years of the last menstrual period) and 57 postmenopausal women. Tumour material, removed at mastectomy or biopsy from the primary cancer, was kept at 0°C and immediately transferred to the laboratory. Following removal of tissue for histopathological diagnosis, the remaining material was dissected free of extraneous fat and divided for assays of prostaglandins and steroid receptors.

Measurement ofprostaglandins
Formation of derivatives Tumour samples were weighed and homogenized in ethanol as previously described (Watson et al., 1984). Internal standards (20-ethyl PGF2ot and 20-methyl PGE2) were added and the samples derivatized by oximation and methylation. Further derivatization to the t-butyldimethylsilyl ether was then performed and the resultant sample purified by passage through a Sephadex LH-20 column and analysed by glc-ms.
Gas chromatographymass spectrometry Samples were analyzed with an Erba Science gas chromatograph coupled through an all-glass jet separator to a V.G. 305 spectrometer as previously described (Watson et al., 1984). For analysis of PGF2x the mass used was m/z 653 and 681 and for PGE2, m/z 666 and 680. The ions measured were the M-57 ions resulting from the loss of a t-butyl radical from the molecular ion. Quantitation was achieved by comparing the areas of the sample peak with those of the corresponding standards. Procedural losses (2040% ) were corrected by monitoring the recovery of the internal standards. For intraassay precision the coefficient of variation was 13%, (n=34); and values for interassay precision were 18 and 21% (n=9) for PGF2a and PGE2 respectively Oestrogen receptors Level of oestrogen receptors was determined by saturation analysis (Hawkins et al., 1975). Tumour cytosol was incubated overnight at 4"C with (2,4,6,7) [3H] 17 1ioestradiol (100 Ci mmol-1). Free and bound steroid were separated by addition of dextran-coated charcoal. Following centrifugation the bound fraction (in the supernatant) was measured by liquid scintillation counting. Quantitation of receptors was determined by Scatchard analysis (Scatchard, 1949). Values in excess of 5fmolmg-' cytosol protein were designated receptor positive.

Statistical analysis
As the distribution of values for both PGF2a and PGE2 was skewed, non-parametric tests (i.e. Wilcoxon's rank test and Spearman's rank correlation) were used throughout. Statistical differences are given by P values as indicated in the text except for those not reaching significance (P>0.05).

Results
The range of values for PGE2 and PGF2 c was respectively, 6 to 977ngg-' tissue (median 75) and 2 to 817ngg-I tissue (median 56) in the 78 tumours ( Figure 1). There was a highly significant correlation between amounts of PGE2 and PGF2a (P<0.001) as previously reported (Watson et al., 1984).
In order to determine if tumour levels of PGE2 and PGF2 relate to prognosis in patients with early disease, the prostaglandins were examined in relation to oestrogen Lymph node involvement receptor status, lymph node involvement, tumour T stage (assessed according to UICC TNM classification) and disease-free interval. Oestrogen receptors were present in 60 of the 78 tumours (77%). The median value of both PGE2 and PGF2 x was higher in tumours with oestrogen receptors but the difference was not significant from the receptor-negative subgroup ( Figure 2). Of 77 patients in whom lymph nodes were biopsied 54 patients (70%) had histologically involved nodes. There was, however, no significant difference in either tumour PGE2 or PGF2 levels between the lymph node positive and negative groups (Figure 3). The number of patients staged as T1 to T4 was respectively 6, 49, 1 1 and 12. There was no significant difference in levels between these groups (Figure 4), although the median value tended to increase with advancing stage.
In 44 patients at least 30 months had elapsed since their initial biopsy or mastectomy. During this period 14 patients presented with recurrent disease while the remaining 30 appeared disease-free. There was, however, no significant difference in either PGE2 or PGF2a levels between these two subgroups of patients ( Figure 5). No significant difference in prostaglandin levels was detected between tumours which developed subsequent recurrences at different sites (i.e. local, bone or visceral), but numbers are too small for meaningful analysis (Table I). Considerable attention has been given to the possible role of prostaglandins in the natural history of breast cancer, largely as a result of reports that breast tumours may contain elevated amounts of prostaglandin-like material (Bennett et al., 1977). Since prostaglandins possess both osteolytic (Klein & Raisz, 1970) and haemodynamic properties (Moncada & Vane, 1980), local production of prostaglandins by tumour cells might aid subsequent spread. Fiu 5 Levels of prostaglandins in tumours which did not recur within 30 months of initial treatment (NR) and in tumours which recurred within this time (R). Lines 'represent median results. No significant difference between the groups by Wilcoxon rank test.  (77) 43-89 (74) Initial results from one group (Bennett et al.. 1977). using bioassay techniques to measure prostaglandins. provided confirmatory evidence for this theory. patients with tumours possessing high basal or synthesized levels of prostaglandins being more likely to present with skeletal metastases and having shorter post-surgery survival times (Bennett et al.. 1979). However. more recent results from other workers have been conflicting and have not necessarily found that elevated tumour prostaglandins are related to poor prognosis.
Thus. studies of prostaglandin synthesis in microsomal preparations of breast cancers showed an increased production both in tumours with a poor prognosis on account of nodal involvement or the absence of steroid receptors. and in those with a better prognosis due to a low T-stage (Rolland et al.. 1980).
Furthermore. there are studies in which high prostaglandin levels are consistently associated with good prognostic factors. For example. Vergote et al. (1985) found higher PGF,i levels in tumours with steroid receptors and in those from women without nodal metastases. Whilst this positive correlation with steroid receptors has been confirmed by Campbell et al. (1981). Fulton et al. (1982). Karmali et al. (1983) and Watson and Chuah (1985). others (Wilson et al.. 1980: Watson et al.. 1984 found no significant difference in prostaglandin levels between oestrogen receptor positive and negative tumours. There are. therefore, wide discrepancies in reports from different groups which could be attributable to several vanrable factors. For example, Bennett's studies (1977). are on tissues from several London hospitals. This would be expected to exacerbate variability associated with the time interval between biopsy and extraction and there may be differences in the degree of tissue trauma and operational procedures which might affect the levels of prostaglandins. Enzymes associated with prostaglandin synthesis are particularly labile (Egan et al.. 1978) and it is essential to minimise delay and trauma in tissue processing. Pathological assessment and clinical staging can also vary between centres and so may introduce further inconsistencies.
Previous studies have also used a variety of methods by which to measure prostaglandins and many have employed bioassay or radioimmunoassay techniques, which do not positively identify individual prostaglandins. Bioassay measures only 'prostaglandin-like material', and the accuracy of radioimmunoassay depends on the specificity of antibodies used. Furthermore, different preparations of tumour tissue have been used for prostaglandin estimation. Some workers have employed microsomal preparations (Rolland et al.. 1980). Others have used crude tumour homogenates (Bennet et al., 1977), either extracting directly with ethanol to measure 'basal' levels or indirectly from an aqueous solution after incubation with endogenous precursor or added arachidonic acid to determine 'synthesized' levels.
Against this confused background, it is clear that the addition of a further uncontrolled study is unlikely to swing the balance decisively in favour of prostaglandins being either a poor or favourable prognostic sign. It is, therefore. worth emphasizing the characteristics of the present study which commend it. Firstly. all the tumours are derived from patients attending a single breast unit. This means that (a) all patients were staged. treated and followed-up according to strict and uniform protocols; (b) all the tumours were routinely collected from the same operating theatre and were subject to similar transport procedures. The problem of variations in handling time due to the constraints of clinical and pathological demands have thus been minimised although not eliminated: (c) prostaglandins have been measured by glc-ms. the most definitive method of identification presently available. We elected to measure 'basal' levels of prostaglandins for the reasons previously explained (Watson et al.. 1984).
Using these methods. levels of tumour PGE, and PGF,x were not significantly related to tumour oestrogen receptor status. lymph node involvement. tumour size, or. in a subset of patients. disease-free interval. This study, therefore. provides no definitive evidence that prostaglandins are related to already established prognostic factors. The absence of a correlation with early tumour recurrence would also appear to exclude a role for the prostaglandins as an independent prognostic parameter. Our study does not exclude the possibility that prostaglandins are involved in the later stages of the natural history of the disease. but this can be determined only by extended patient follow-up.
Measurements in the present study have also been confined to PGE, and PGF, o but many physiological and pathological effects formerly associated with classical prostaglandins may be attributable to the action of other oxygenated metabolites of arachidonic acid. It may. therefore. be pertinent that in a recent study (Karmali et al.. 1983) thromboxane B2 was the only arachidonate metabolite to show a significant relationship with tumour size. positive lymph nodes and distant metastases.
In conclusion, while greater efforts can be made to control the numerous non-specific variables involved in the measurement of prostaglandin levels in breast tumours. it is doubtful whether such studies on classical prostaglandins. PGE2 and PGF2:x, are likely to provide useful information on the dissemination and natural history of breast cancer.