Overall, the patterns of expression of p53 and Ki-67 in the IBMFS marrows resembled the patterns in AML and MDS, and were higher in the inherited syndromes and the sporadic hematologic malignancies than in acquired aplastic anemia or normal control marrows.
p53 was not expressed in AA or normal bone marrows, as expected, and the previously reported increased expression in sporadic AML and MDS was confirmed. As a group, the IBMFS marrows also overexpressed p53 in frequencies similar to those in the sporadic conditions, and this overexpression was not associated specifically with MDS morphology in the IBMFS. In addition, the frequency of overexpression of p53 in patients with an IBMFS who expressed p53 was not only the same as or higher than the proportion of those with sporadic MDS and AML, but also the proportion of p53 positive cells in the IBMFS patients who did express p53 was higher than in the sporadic controls. The DC and SDS marrows in which p53 was expressed had the highest proportions of positive cells in any group.
The proportion of patients with an IBMFS who overexpressed Ki-67 was similar to the proportion with sporadic AML, and both were higher than those with MDS. Amongst the overexpressors of Ki-67, substantially more cells were positive in the IBMFS marrows than in the sporadic marrows.
Thus, the patterns of expression of p53 and Ki-67 in the four main types of IBMFS that we examined resemble the patterns described in the literature and confirmed here for MDS and AML. Although the frequency of MDS morphology was low (9 to 30%) among our IBMFS marrows, the frequency of overexpression of these two cell cycle markers ranged from 50 to 89%, and thus overexpression was clearly present in high proportions of patients who had normal marrow morphology. The frequency of overexpression was generally similar in all four of the syndromes. The proportion of positive cells among the expressing marrows was somewhat higher in IBMFS than in sporadic AML and MDS. SDS and DC overexpressors had slightly more p53 positive cells than did FA and DBA, but the numbers of patients are small.
The results with survivin were unexpected. First, in contrast with previous reports, only 1 or 2 of the patients with sporadic disorders had any positive cells. Second, and related to the low expression in the controls, more than 75% of the IBMFS marrows had survivin-positive cells, and the proportions of positive cells in the overexpressors ranged from 30 to 95%, much higher than the 30-40% positive in the sporadic controls. This leads to the conclusion that, at least with our method, a high level of expression of survivin protein in a bone marrow biopsy may be helpful in distinguishing an inherited from an acquired bone marrow failure disorder. While it will not lead to specific syndromic diagnoses, it may indicate that an IBMFS work-up should be initiated. The results of a survivin immunohistochemistry assay can be available much more quickly than the results of chromosome breakage analysis for FA, telomere length for DC, and specific surrogate tests for DBA and SDS [2
Our observations are in agreement with the published literature that indicates overexpression of p53 and Ki-67 in MDS/AML [5
]; however, the close resemblance in overexpression of these markers between IBMFS and MDS/AML has not been reported previously. Similarities in the rate of overexpression of p53 and Ki-67 between IBMFS and MDS/AML may suggest a common cell cycle deregulation mechanism that is responsible for the preleukemic and leukemic processes in these marrow disorders. Moreover, the lack of association between p53 and Ki-67 in DBA and the negative association in DC in the face of a positive association of these two markers in AML and in FA and SDS, which have a significantly higher risk of MDS/AML [2
], may be due to ineffective p53-dependent apoptosis or a cellular attempt to compensate by intensifying the apoptosis signal to balance out cell proliferation as indicated by Ki-67.
Unexpectedly, we found a significantly higher rate of overexpression of survivin in IBMFS compared with AML, MDS-hi and MDS-lo, and no difference in the expression of survivin in AML and MDS compared with normal. While the literature indicates overexpression of survivin in MDS/AML [12
], a gradual increase followed by a rapid decrease in survivin expression has been described in association with MDS progression to AML in a limited number of patients [15
]. While none of the acquired bone marrow disorder groups showed any correlation between the overexpression of survivin and Ki-67, we observed a strong positive correlation in the IBMFS marrow samples (p <0.008, r2
= 0.5-0.7). This observation is logical since survivin promotes cell proliferation and Ki-67 reflects its occurrence; however, the time sequence of overexpression of these markers was not examined in our study. A decrease in the expression of survivin in the immediate preleukemic or early leukemic period cannot be excluded due to the lack of samples from patients with IBMFS who developed AML. While DC showed a negative association between p53 and survivin (p = 0.02, r2
= 0.6) and FA and DBA showed no significant association (p >0.06), only SDS showed a positive association (p = 0.01, r2
= 0.36). This makes the theory of a reactive survivin elevation in an attempt by the marrow to overcome the p53-dependent apoptosis less likely and provides more support to the previous hypothesis of an ineffective p53-mediated apoptotic pathway in IBMFS. Survivin may provide the missing link for cell salvage from apoptosis by counteracting the p53 overexpression, rendering ineffective its apoptosis-mediated pathway. However, it is not clear from our study whether survivin overexpression is an inherent mechanism in IBMFS or is temporarily activated to maximize the proliferative activity of a smaller pool of marrow cells. Follow-up of IBMFS patients using these immunohistochemical stains may provide additional information regarding the risk of developing further bone marrow complications.
Our analysis included a relatively large number of samples from each disorder, which were analyzed in a single laboratory. The sample size and the standardized, masked analysis represent strengths of our study; however, the study is currently limited by its cross-sectional design. We have subsequent analyses on only half the patients; 11 had bone marrow transplantation for aplastic anemia, and 8 died from complications of their aplastic anemia. The median follow-up period is only two years (range 0.5 to 7 years), and none of the patients have progressed to full-blown MDS or AML. This study is the first systematic examination of cell cycle marker expression in all of the IBMFS, and we identified overexpression patterns which resemble those seen in patients with primary MDS or AML, even in patients with an IBMFS who did not have MDS/AML. A unique finding was the very high expression of survivin in the IBMFS and not in any of the acquired conditions.
Much longer follow-up and serial analyses of the expression of cell cycle regulation markers in IBMFS marrows are required to determine whether these markers serve only to distinguish inherited from acquired marrow disorders, or whether they identify patients with IBMFS who are at imminent risk of evolving into MDS and/or AML.