To the Editor: Calin et al. (Oct. 27 issue)1 report on genomicmutations within microRNA loci in patients with chronic lymphocyticleukemia (CLL). We believe that the frequency with which suchmutations can be found was not accurately estimated and thatsome of the cell-based data were unsound. Among 69 samples ofCLL cells, in which one copy of chromosome 13q was lost, wedid not identify mutations within a 581-bp segment encompassingthe miR-15a–16-1 gene. We also consider it very surprisingthat a single substitution outside the pri-miR-16-1 hairpinabolished the expression of both miR-15a and miR-16-1 as presentedin Figure 2E of the original article. Figure 2E is inconclusiveand mislabeled. It appears that miR-15a (mislabeled as U6, whichis a 100-nucleotide small nuclear RNA) is present in all samplesand that miR-16-1 is absent from even the control 293 cells.MiR-15a represents about 5 percent, and miR-16-1 about 10 percent,of the total microRNA content of the 293 cells. It remains unexplainedwhy miR-15a was detected in the left portion of Figure 2E butmiR-16-1 was not detected in the right portion, showing thecontrol human embryonic kidney 293 cells.
Tom Tuschl, Ph.D. Rockefeller University New York, NY 10021
References
Calin GA, Ferracin M, Cimmino A, et al. A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 2005;353:1793-1801. [Free Full Text]
To the Editor: Calin et al. found a significant associationbetween a microRNA expression signature and ZAP-70 expression,mutational status of the immunoglobulin heavy-chain variable-region(IgVH) gene, and time to initial treatment in patients withCLL. They suggest that microRNA expression can be included asa prognostic marker in CLL. This proposal is premature, sincethe microRNA signature appears to be a surrogate for high ZAP-70expression and unmutated IgVH, which are known to be markersof a poor prognosis in patients with CLL.1,2,3 Was a multivariateanalysis done to show that microRNA is an independent prognosticfactor? Furthermore, it is not clear that the list of microRNAgenes that predict time to treatment completely overlaps withthe microRNAs that are associated with ZAP-70 expression andIgVH mutational status (only five are the same), especiallysince the chip used to detect microRNA can discriminate betweensimilar isoforms of microRNAs. Finally, it is not clear whetherthe set of nine microRNAs that predict progression was validatedin the independent set of 50 patients with CLL.
Wee J. Chng, M.D. National University Hospital Singapore 119074 chngwj{at}nuh.com.sg
References
Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;351:893-901. [Free Full Text]
Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764-1775. [Free Full Text]
Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848-1854. [Free Full Text]
The authors reply: In our article, we reported the sequencesof 42 microRNA genes in 75 patients with CLL and found mutationsin 5 of the 42 genes in 11 patients. For this reason, we aresurprised that Borkhardt et al. questioned our estimates onthe basis of their failure to find mutations in the miR-15a–miR-16-1transcript in 69 cases of CLL. That Borkhardt et al. did notfind this rare germ-line mutation, which we identified in only2 of 75 patients, is not unexpected. Moreover, the frequencyof germ-line mutations may vary in different populations,1 andone of our patients with a germ-line mutation in miR-15a–miR-16-1had familial CLL. The reduction in gene expression induced bythis mutation was reproduced in two independent experimentsand by a quantitative reverse-transcriptase–polymerase-chain-reactionassay. The mechanism responsible for this effect remains tobe identified. Borkhardt et al. are correct that Figure 2E ismislabeled. The headings were reversed; the left portion shouldread "miR-16-1," and the right portion should be labeled "miR-15a."
Contrary to the assertion by Chng, our extended signature, associatedwith ZAP-70 expression (presented in the Supplementary Appendixto the article) and composed of 19 microRNAs, includes all 9microRNAs that predict time to treatment (and not 5, as statedby Chng). We did not conclude that microRNA expression shouldsubstitute for ZAP-70 expression and IgVH mutation status asa prognostic marker in cases of CLL but, rather, that it isa new prognostic marker. Whether it is an independent prognosticfactor has yet to be determined. Finally, since we had no survivaldata for the test set of 50 samples of CLL, we restricted ouranalyses to cases with data on ZAP-70.
George A. Calin, M.D., Ph.D. Amelia Cimmino, M.D., Ph.D. Carlo M. Croce, M.D. Ohio State University Columbus, OH 43210 carlo.croce{at}osumc.edu
References
Liede A, Karlan BY, Narod SA. Cancer risks for male carriers of germline mutations in BRCA1 or BRCA2: a review of the literature. J Clin Oncol 2004;22:735-742. [Free Full Text]
Ji, R., Cheng, Y., Yue, J., Yang, J., Liu, X., Chen, H., Dean, D. B., Zhang, C.
(2007). MicroRNA Expression Signature and Antisense-Mediated Depletion Reveal an Essential Role of MicroRNA in Vascular Neointimal Lesion Formation. Circ. Res.
100: 1579-1588
[Abstract][Full Text]
Cheng, Y., Ji, R., Yue, J., Yang, J., Liu, X., Chen, H., Dean, D. B., Zhang, C.
(2007). MicroRNAs Are Aberrantly Expressed in Hypertrophic Heart: Do They Play a Role in Cardiac Hypertrophy?. Am. J. Pathol.
170: 1831-1840
[Abstract][Full Text]
Linsley, P. S., Schelter, J., Burchard, J., Kibukawa, M., Martin, M. M., Bartz, S. R., Johnson, J. M., Cummins, J. M., Raymond, C. K., Dai, H., Chau, N., Cleary, M., Jackson, A. L., Carleton, M., Lim, L.
(2007). Transcripts Targeted by the MicroRNA-16 Family Cooperatively Regulate Cell Cycle Progression. Mol. Cell. Biol.
27: 2240-2252
[Abstract][Full Text]
Diederichs, S., Haber, D. A.
(2006). Sequence Variations of MicroRNAs in Human Cancer: Alterations in Predicted Secondary Structure Do Not Affect Processing.. Cancer Res.
66: 6097-6104
[Abstract][Full Text]
Previous
