The oligosaccharides extracted from the different breast cancer specimens were assessed further by comparing the number of sialylated species separated on anion-exchange chromatography, as shown in figure 3.4.
Sialylated oligosaccharides elute from the anion-exchange column according to their size, the number of acidic residues attached and depending on the
anomericity o f the bond between the sialic acid and adjacent monosaccharide (a2- 3, a2-6 or a2-8) (Guile et al., 1994). The specimens from the short-term survival breast cancer patients showed a statistically significant decrease in diversity o f charged oligosaccharide structures compared with the specimens from the long term survivor patients. Student t-test p=0.006.
Figure 3.3 Sialylated oligosaccharides from long-term and short-term breast cancer survivors. Mean values +/- standard error and Student t test P value
indicated. (L> T3
1
C/5I
1
i
80 1 60 40p=0.052
o o o O 8 long-term survivors short-term survivorsIn each of the populations the distribution of the relative quantities of
oligosaccharides were found to show a normal distribution about the mean and a paired Student t-test was used to compare the relative amounts of sialylated oligosaccharides in the two populations.
There was a trend towards significantly decreased amounts of sialylated oligosaccharides in the short-term survival group (p = 0.052). This was due to four of the ten short-term survivor specimens having much reduced levels of sialylated oligosaccharides compared with the specimens from the long-term survivors. No apparent difference in the histological or clinical features, such as extent of lymph node métastasés, was identified to account for the reduced levels of sialylated structures in the four short-term survival specimens.
Figure 3 .4. Numbers of sialylated oligosaccharides separated on anion- exchange chromatography from breast cancers of short-term and long-term survivors. Mean values +/- standard error and Student / test P value indicated.
25 1
I
I
I
I
o o o o1
short-term survivors long-term survivorsThere was an overlap between the number of oligosaccharides resolved on anion- exchange chromatography from the short-term and long-term survivor specimens. In the long-term survivor specimens, there were two specimens for which few sialylated structures were resolved, in the short-term survivors there were two specimens for which many sialylated oligosaccharides were resolved on anion- exchange chromatography. The reason why this variation occurs is not obvious, examination of the H+E stained section does not provide any obvious
explanation(s), nor do the clinical features that were recorded for the specimens.
3.5 Results : neutral oligosaccharides.
Where possible, the neutral oligosaccharides from a number o f the specimens were evaluated by separation on a BioGel P4 gel permeation chromatography column. Only a few of the samples could be run in this way, since some o f the specimens had insufficient oligosaccharide present for detection following gel
permeation separation. Of those samples run, the analysis indicated an absence of larger oligosaccharides in the short-term survival specimens, figure g.5. Also, an increase in the relative quantities of small neutral glycans was observed in the short-term survival specimens. One of the structures over expressed in the short term survival specimens, figure 3.6, was analysed further on a CarboPak PAIOO column in monosaccharide mode. The structure over-expressed in the short-term survival specimens was found to elute at the same time as GalNAc, figure 3.7.
Figure 3.5 Neutral oligosaccharides from long-term survival (blue) and short term survival (red) cancer specimens, separated on a BioGel P4 column fitted to a
GlvcoMat) 2000. Elution positions of hvdrolvsate of dextran in glucose units (GU) shown. 10 0 -1 60-1 STR3453-78 8 0 - 50 - 6 0 - 4 0 - 3 0 - 20 4-
bme (minute 0 tone (mmutes)
BAR3862-79 FEN3204-79 4 0 - 3 0 - 3 0 - cm t (mmutes) ame (minutes) 1 1 1
Figure 3.6 BioGel P4 séparation of oligosaccharides from breast cancer specimens of a long-term survival (blue) and a short-term survival (red) breast
cancer patient. The peak over-expressed in the short-term survival patient is shown by the arrow.
100 80 GO 40 V . 20 40 30 v o l (m l) 20
Figure 3.7 Analysis of a monosaccharide over-expressed in the short-term survival breast cancer specimen on a CarboPak PAIOO column. The elution
positions of 2AB-labelled GlcNAc and GalNAc are indicated.
G l c N A c G a l N A c ; i O 07 a 0 6 0.05 f O 04 o> O 03 8 0. 02 0.01 0 OO -0 . 0 1 1 0 t i m e ( m l n )
J. 6 Discussion o f pilot study.
We have used techniques, developed for the extraction of oligosaccharides from archival breast cancer specimens, to compare oligosaccharides from primary breast cancers of patients who did / did not develop clinical métastasés. The primary breast cancers were from post-menopausal patients presenting in the clinic with similar grade and stage disease, who underwent similar surgical and post-operative therapies.
Various differences have been identified in the oligosaccharides extracted from the ‘metastatic’ compared with the ‘non-metastatic’ breast cancers. In particular, a reduction in the amount of sialylated oligosaccharides, as a percentage of the total oligosaccharide pool was found in the short-term survival specimens. This does not appear to be due to a failure of sialylation o f neutral oligosaccharides, since the relative quantities of neutral glycans were not increased in the specimens from the short-term survival compared with the long-term survival patients.
Also, a significant reduction in the number of different charged oligosaccharide structures was observed in the specimens from the short-term compared with long-term survival patients (p=0.006). Reduced numbers of neutral
oligosaccharide structures were also seen in the short-term, compared with the long-term, survival patients. Finally, the short-term survival patients showed an over-expression of the monosaccharide GalNAc.
The cellular events which might lead to the production of different glycoforms in the breast cancers with different clinical behaviours have not been investigated in this study. The results, of decreased sialylated glycans and decreased numbers of sialylated and neutral structures in the short-term survival specimens, both suggest that an overall failure of glycosylation may be occurring. Possible reasons for the differences in the oligosaccharides extracted from the short-term survivor
specimens include the inappropriate expression of glycosyltransferase enzymes in the Golgi, reduced levels o f donor sugars and faster turnover o f cellular proteins. Various reports suggest that there are differences in the levels and activities of glycosyltransferases in cancer cells, for example, increased levels and activity of sialyltransferase (CMP-sialic acid Gaip 1-3GalNAc a3-sialyltransferase) in some breast cancer cell lines resulting in the over-expression of shortened 0-linked oligosaccharides (Brockhausen et al., 1995). To date, however, there have not
been any reports of cancer-associated changes in glycosyltransferases which would explain the increased expression of GalNAc observed in poor prognosis breast cancers, but as the mode of operation of the enzymes o f glycosylation becomes better understood, mechanisms which lead to increased GalNAc expression on proteins may become apparent.
The results of this study correlate with some reports of altered glycosylation in breast cancer. Brockhausen et al have reported decreased branching and reduced diversity o f sialylated 0-linked oligosaccharides in breast cancer cell lines, as noted above (Brockhausen et al., 1995), but this has not been reported to be associated with the formation of métastasés. Such associations may need to be verified by clinical studies similar to this one. The increased level of GalNAc extracted from the specimens of the short-term survival patients also correlates with reports of increased Tn antigen expression (Springer, 1989) and HP A lectin binding (Brooks and Leathem, 1995a; Brooks and Leathem, 1991) in poor prognosis breast cancers. We did not find any association between increased p i- 6GlcNAc branching and polylactosamine expression in the metastatic breast cancers, despite reports of its importance in the formation of métastasés in an animal model (Dennis g/ a/., 1987).
The formation of métastasés is undoubtedly a multi step event that is dependent on many factors, as described in section 1.2.6. Nevertheless, the differences
observed between the oligosaccharides extracted from the metastatic compared with non-metastatic breast cancers suggest that particular changes in glycosylation o f proteins are associated with, and may be advantageous for, the successful development of métastasés. There are various ways in which changes in
glycosylation may affect the function and recognition of secreted and cell surface proteins, discussed in section 1.3.1. Some changes in glycosylation of proteins might confer advantages to a cell during the process of metastasis, for example by affecting cell adhesion, section 1.2.5.4, or changing the immune recognition o f cancer cells, section 6.6. To understand better which oligosaccharides are important in primary breast cancer and their possible role in the metastatic process, a larger series o f specimens needed to be examined and multivariant analysis performed. This has been addressed in a larger retrospective study o f 76 patients as described in chapter 5.
Chapter 4
Oligosaccharides associated with poor prognosis determined by Helix pomatia agglutinin (HPA) lectin binding to breast cancer cell lines.