There is a considerable molecular
There is a considerable molecular genetic heterogeneity within ES/PNET. As mentioned above, either or can rearrange with in these gene fusions. Furthermore, for either gene fusion, additional heterogeneity stems from the location of the genomic breakpoints of the translocation, resulting in different combinations of exons from and (or ). The most common fusion joins Dihydro-β-erythroidine hydrobromide receptor 7 in frame with exon 6 (type 1 fusion). There are at least 12 other - types, and at least 4 types of - fusion types described. We and others have previously reported that among patients with - fusions, those having type 1 transcripts have a better outcome regardless of conventional prognostic factors such as stage, age, or tumor location.
The biological mechanisms underlying this reproducible and significant association of fusion structure and clinical behavior are unclear. As a first clue to how these variables may be linked, we have recently found that the - type 1 fusion may encode a transcription factor with lower transactivation efficiency than other fusion types. Because studies using various approaches to inhibit EWS-FLI1 function have shown that it is a critical determinant of proliferation in ES/PNET cell lines,, , we have now sought to determine whether the observed covariation of structure, function, and clinical course extends to tumor cell kinetic parameters such as proliferative rate and apoptosis. We have also examined expression of the receptor for insulin-like growth factor 1 (IGF-1R), because there is evidence from several groups implicating it in autocrine or paracrine control of ES/PNET growth., , , The results of our analysis, described below, are consistent with the notion that type 1 EWS-FLI1, functionally a weaker transcription factor, may result in lesser activation of direct or indirect target genes, possibly including IGF-1R, controlling proliferative rate in ES/PNET.
Materials and Methods
Results Immunoreactivity for Ki-67 obtained with the MIB1 antibody had a nuclear pattern with strong nucleolar staining (Figure 1). Immunostaining for Ki-67 was most frequently seen in nuclei of dark cells, that seldom showed TUNEL reactivity. In ES/PNET, cells with hyperchromatic nuclei (dark cells. are usually seen along with cells having clear nuclei. They have traditionally been regarded as apoptotic cells. Interestingly, nuclei reactive with the TUNEL technique were usually those of clear cells, whereas those of dark cells appeared positive less often. This pattern of TUNEL reactivity in some clear cells could represent an early stage of apoptosis, because the characteristic morphological appearance of apoptotic nuclei is a later event in the apoptotic process. Membranous staining was seen with the antibody to IGF-1R (Figure 2). Most cases showed widespread IGF-1R immunoreactivity and all cases contained at least some positive cells. The mean and median percentages of tumor cells positive for these markers were respectively as follows: Ki-67 (n = 85) 21% and 14%, IGF-1R (n = 78) 73% and 80%, and TUNEL (n = 66) 5% and 2%. There was a significant positive correlation between proliferative index as assessed by immunostaining for Ki-67, and IGF-1R immunoreactivity (Spearman correlation coefficient, r = 0.23; P = 0.04). All cases with Ki-67 immunoreactivity in >20% of tumor cells had IGF-1R expression in 50% or more of cells (Figure 3). Conversely, all cases with <40% IGF-1R-positive cells had a Ki-67 proliferative index of 10% or less. There were 72 tumors with EWS-FLI1 transcripts: 44 type 1 fusions and 28 grouped into the non-type 1 group. Fourteen tumors had EWS-ERG fusions. Patients having EWS-FLI1 fusions had similar age, stage at diagnosis, and tumor volume with respect to those bearing EWS-ERG fusion genes (results not shown). In the group of tumors with EWS-FLI1 transcripts, those having type 1 fusions had a lower immunoreactivity for Ki-67 (15%; P = 0.049), and for IGF-1R (65%; P = 0.015) than tumors with other EWS-FLI1 fusion types (24% and 82%, respectively) by Mann-Whitney test (Table 1). In aggregate, tumors with EWS-FLI1 transcripts had lower values for Ki-67 (P = 0.01) than those with EWS-ERG fusions by Mann-Whitney test (Table 1).