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Letter to the Editor
Comparable expression and phosphorylation of the retinoblastoma protein in Merkel cell polyoma virus positive and negative Merkel cell carcinoma
Article first published online: 27 JUL 2009
Copyright © 2009 UICC
International Journal of Cancer
Volume 126, Issue 3, pages 796–798, 1 February 2010
How to Cite
Houben, R., Schrama, D., Alb, M., Pföhler, C., Trefzer, U., Ugurel, S. and Becker, J. C. (2010), Comparable expression and phosphorylation of the retinoblastoma protein in Merkel cell polyoma virus positive and negative Merkel cell carcinoma. Int. J. Cancer, 126: 796–798. doi: 10.1002/ijc.24790
- Issue published online: 8 DEC 2009
- Article first published online: 27 JUL 2009
- Accepted manuscript online: 27 JUL 2009 12:00AM EST
- Manuscript Accepted: 21 JUL 2009
- Manuscript Received: 17 JUL 2009
- Wilhelm-Sander Stiftung. Grant Number: 2007.057.1
It has been demonstrated, that DNA from the Merkel cell polyoma virus (MCV) is monoclonally integrated in the genome of Merkel cell carcinoma (MCC) cells in the majority of tumors.1 In this respect, Bathia et al. recently reported an observation in THE JOURNAL which suggests that two subgroups of MCC can be distinguished on the basis of the abundance of MCV; moreover, these subgroups differed in their expression of cancer related proteins, i.e. the Retinoblastoma protein (RB).2 The authors report that MCV DNA load was less then one copy per 300 cells in 14 of 23 MCC tumors (60%), and that these tumors were characterized also by loss of RB expression. In the remaining samples, which were characterized by high levels of RB expression, the estimated viral load was always higher than 1 copy per 20 cells. Noteworthy, only in two cases viral load was higher than 1 copy per 2 cells. In consequence, the authors speculate on a possible mechanism in MCC with a minority of infected cells contributing to transformation of uninfected neighbouring cells by paracrine mechanisms.2 However, the results of Bathia and colleagues are in contrast to another recently published paper describing the viral load in MCV+ MCCs to be generally >1 copy per tumor cell as measured by quantitative PCR.3 Moreover, immunohistological analyses of MCV+ MCCs demonstrated that nearly every tumor cell expressed the MCV large T-antigen (LTA) protein. Confirming this latter report, our own immunohistochemical analysis of MCC tissues confirms this pattern of LTA expression, i.e. virtually every tumor cell of MCV+ MCCs is expressing LTA protein (Fig. 1a). Moreover, we analyzed genomic DNA purified from 50 MCC tissue samples for the presence of MCV by Real time PCR. Relative quantification of the samples was calculated by the ΔΔCt method normalized to the repetitive DNA elements LINE1; genomic DNA of a MCC cell line that harbours at least two concatemerized copies of the MCV genome in every cell served as calibrator which allowed to approximate the minimal copy number per cell in the tissues. To this end, MCV DNA was undetectable in only 7 of the 50 samples (14%). In those samples with detectable MCV DNA the median of the estimated minimal copy numbers is approximately 1 copy per cell (Fig. 1b). Indeed, 63% of the MCV positive cases (27 samples) demonstrate minimal copy numbers higher than 0.5 copies/cell. Even some of the values below 0.5 may still be in accordance with 1 MCV copy per tumor cell as some of the tissue samples harbour high proportions of non tumour cells. In only 7 MCV+ samples we estimated a MCV copy number of less than 1 per 20 cells; thus, we can not confirm the findings of Bhatia et al. with respect to the viral load being very low in the majority of MCC cases.
In addition to the differences in virus copy number, we obtained also different results with respect to RB expression; Bhatia and colleagues observed RB negative MCC cases in 60%. In contrast, we could not detect loss of RB expression in a substantial proportion of MCC cases. In fact, we detected RB protein in all samples in virtually all tumor cells when applying immunohistochemical analysis to 50 different paraffin-embedded MCC tumor tissues (Fig. 2a). This series included also 7 cases negatively tested for MCV DNA and 7 cases with relatively low approximated copy numbers indicating that viral abundance in MCC tissues is not correlated with RB expression (Fig. 1b). The presence of RB in the vast majority of MCC tissues was further substantiated by use of a phospho-RB specific antibody which detected the protein in immunohistochemical analysis in a subset of 12 MCC samples (Fig. 2a). Notably, the staining pattern with only some of the tumor cells displaying phosphorylation of RB is consistent with RB phosphorylation being a cell cycle dependent event. Indeed, this has been described both, for non virus-related but also for T-antigen transformed cells with the difference that in the latter the phosphorylation occurs after rather than before entry into the S phase.4 Interestingly, phospho-RB could be detected despite the fact that considerable levels of the CDK inhibitor p16INK4a were expressed in all 50 analyzed MCC tissues (Fig. 2a). Cell cycle dependent RB phosphorylation in the presence of high levels of p16INK4a was also confirmed in three different MCV− MCC cell lines. These cell lines increased phospho-RB upon release from cell cycle arrest (Fig. 2b).
These different observations may be explained by the different geographical localisation of population from whom the MCC tumors were derived. Indeed, Garneski and colleagues investigated the presence of MCV DNA in 16 and 21 formalin-fixed MCC tissues from patients in North America and Australia, respectively.5 Using real-time PCR with primers sets designed to amplify regions of the MCV genome corresponding to the small and large T-antigens, MCV DNA was detected in 43% of the total 37 MCC tissues. When samples from North America and Australia were considered separately, the prevalence of MCV DNA in MCC tissues was greater for those samples obtained from North America (69%) as compared to Australia (24%) (p = 0.009).
In conclusion, our analysis demonstrates that RB expression is a general feature of both MCV+ and MCV− MCC cells. Inactivation of the RB pathway is proposed to occur in virtually all tumors; this may be achieved in MCV+ MCC cells by expression of the MCV LTA, which is suspected to inactivate RB.6 For MCV− MCCs the mechanism is less clear. Loss of p16INK4a, which binds to CDK4 and CDK6 and thereby prevents phosphorylation of RB at the G1/S transition, is a critical RB pathway inactivating event in several tumors. However, our data suggest that in MCV− MCC neither this nor loss of RB expression take place. Instead, the observed cell cycle dependent phosphorylation of RB in the presence of high levels of p16INK4a indicate a similar RB inactivating event in both MCV+ and MCV− MCC. However, for the MCV− cases this event is to date completely unclear, but it is tempting to speculate that it may be caused also by a virus.
We highly appreciate that Patrick Moore made his LTA specific antibody—prior to its publication—available to us. Roland Houben was supported by the Wilhelm-Sander Stiftung (grant 2007.057.1).
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Roland Houben, David Schrama, Miriam Alb, Claudia Pföhler, Uwe Trefzer, Selma Ugurel, Jürgen C. Becker.