Routine histopathological examination of pancreases showed no significant findings in normal autopsy controls or patients with gastrinoma, other than mild focal fibrosis in the latter. Samples of pancreases from patients with adenocarcinoma and chronic pancreatitis had varying degrees of perilobular fibrosis, ductular proliferation or hyperplasia, and acinar atrophy that was heterogeneous between lobules (Fig. 1). Islet inflammation (insulitis) was not present in any sample as indicated by the lack of several mononuclear cells surrounding or within the islet by AF-H&E staining. Insulin staining by acinar region was variable within patients and was recognised in islets of varying sizes, small clusters and single cells (Figs 1, 2). Insulin-positive cells were infrequently observed within the ductular epithelium (immunostained with cytokeratin 7) in all groups (Fig. 2).
As previous studies have suggested that beta cell mass may be influenced by a variety of pathological conditions, including those providing the focus for this report, we considered it important to address the issue of fractional insulin area. Fractional insulin area provides an assessment of the proportion of pancreatic cells that are insulin-positive cells, as an indicator of beta cell mass [21, 25–27]. Using this well-accepted method of analysis, very limited differences in fractional insulin area were noted amongst the five study groups using immunohistochemistry (1.4 ± 0.1% autopsy control, 1.1 ± 0.1% no pancreatitis, 2.0 ± 0.2% mild pancreatitis, 1.8 ± 0.7% severe pancreatitis, 1.5 ± 0.2% gastrinoma). Indeed, the only comparison within the five study groups reaching statistical significance was that of patients with no pancreatitis vs those with severe pancreatitis, using the immunofluorescence field of view measurements, where a significantly increased fractional area was noted in the latter group (3.2 ± 0.5% vs 5.9 ± 1.4%, respectively, p < 0.05). Fractional insulin area in the samples from autopsy patients was 4.1 ± 0.6%, which was slightly lower than that from patients with mild pancreatitis (5.2 ± 0.9%) or hypergastrinaemia (5.3 ± 1.0%).
The number of insulin-positive cells in islets, clusters or as single cells was quantified and the total number of insulin-positive cells calculated (Fig. 3a). These data represent a total of 20,693 beta cell counts (i.e. 4,451 autopsy control, 3,407 no pancreatitis, 3,731 mild pancreatitis, 4,122 severe pancreatitis, 4,982 gastrinoma). Similar to the fractional insulin area data, total beta cell number was highest in the severe pancreatitis group, although these data were also the most variable (209 ± 89; Fig. 3a). The no pancreatitis group had the lowest total beta cell number (101 ± 13), which was significantly lower than that in the autopsy control (178 ± 35, p = 0.053), mild pancreatitis (173 ± 26, p = 0.027) and gastrinoma (200 ± 45, p = 0.046) groups (Fig. 3a). Islet beta cell number was similar for all groups except the no pancreatitis group, which had a significantly lower number than the mild pancreatitis group (92 ± 13 vs 157 ± 20, respectively, p = 0.020; Fig. 3b). These two groups showed the lowest variability in beta cell count. The number of beta cells in clusters was similar among autopsy controls (15 ± 4) and patients with no (6 ± 1) or mild pancreatitis (10 ± 5; Fig. 3c). The number in these three groups was lower than that in the severe pancreatitis group (45 ± 15, p = 0.051 vs autopsy controls, p = 0.008 vs no pancreatitis, p = 0.057 vs mild pancreatitis). Furthermore, similar to the severe pancreatitis group, the gastrinoma patients had a significantly increased number of beta cells in clusters compared with the no pancreatitis group (27 ± 6, p = 0.005). Strikingly, the largest increase in beta cell count in the severe pancreatitis and gastrinoma groups was observed for single beta cells, with values (21.3 ± 2.2 and 19.6 ± 5.1, respectively) significantly higher (p < 0.005) than those in the control (3.6 ± 0.9) and no (2.4 ± 0.4) or mild (5.2 ± 2.5) pancreatitis study groups (statistical power 99%, α = 0.05, β = 0.80; Fig. 3d).
For assessment of beta cell replication, Ki-67 nuclear staining of insulin-positive cells was used as an index of the cellular proliferation rate. Of the aforementioned 20,693 beta cells analysed, 86 beta cells had Ki-67-positive nuclei (0.42% overall). Beta cell proliferation rate, expressed as either the percentage of total or islet beta cells with Ki-67 nuclear staining, was significantly increased (p < 0.005) in the severe pancreatitis group compared with all other groups (Fig. 4). The beta cell proliferation rate within clusters was similar for all groups (0.2 ± 0.1% overall; Fig. 4). Single beta cells showed highly variable proliferation rates; the rate was highest in the severe pancreatitis group (3.4 ± 1.7%) but there were no significant differences between groups (Fig. 4). As age has been reported to influence the beta cell replication rate , the ages of the different patient groups were analysed for statistical differences; none were found (autopsy control 43 ± 8 years [n = 5], no pancreatitis 57 ± 7 years [n = 6], mild pancreatitis 46 ± 8 years [n = 4], severe pancreatitis 62 ± 7 years [n = 4], gastrinoma 44 ± 7 years [n = 6]).
Pancreas samples from all study groups were examined for levels of three transcription factors (PDX1, NEUROD1 and NEUROG3) associated with the differentiation of pancreatic endocrine precursors into adult beta cells. Surprisingly, transcription factor levels in islets from the autopsy control samples were lower than those in all other groups (Fig. 5d). These data suggest that pancreatic sample harvesting post-mortem could lead to altered levels of these transcription factors.
PDX1 immunoreactivity was found predominantly in islet cells (Fig. 5a), with low levels observed in pancreatic ducts and acinar cells. Islet levels of PDX1 were significantly higher in both the severe pancreatitis (42 ± 13%, p = 0.009) and gastrinoma (40 ± 14%, p = 0.02) groups than in the no pancreatitis group (3 ± 2%; Fig. 5d). There was virtually no PDX1 present in islet cells of autopsy control patients (0.2 ± 0%; Fig. 5d). Interestingly, patients with mild pancreatitis had intermediate, yet highly variable, islet levels of PDX1 (18% ± 10%).
In contrast to the largely limited islet levels of PDX1, NEUROD1 was observed in islets (Fig. 5b) as well as in ducts and acinar cells. The proportion of NEUROD1-positive islet cells was significantly higher in patients with severe pancreatitis (85% ± 6%, p < 0.02) than in all other patients (Fig. 5d). Islet levels of NEUROD1 were moderately higher in patients with either mild pancreatitis (40 ± 14%) or gastrinoma (34 ± 14%) than in patients without pancreatitis (24 ± 11%; Fig. 5d). Levels of NEUROD1 in ducts and acinar cells were higher in both the severe pancreatitis and gastrinoma groups than in the no or mild pancreatitis groups, but variability precluded demonstration of significant differences (data not shown).
As expected for adult tissues, NEUROG3 levels were lower than PDX1 and NEUROD1 levels (Fig. 5c,d). In addition, the gastrinoma group had the highest islet NEUROG3 levels (18 ± 14%) of all the study groups, although there were no statistically significant differences between any of the groups (Fig. 5d).