Correspondence

Correction

MicroRNA in Chronic Lymphocytic Leukemia

N Engl J Med 2006; 354:524-525February 2, 2006

Article

To the Editor:

Calin et al. (Oct. 27 issue)1 report on genomic mutations within microRNA loci in patients with chronic lymphocytic leukemia (CLL). We believe that the frequency with which such mutations can be found was not accurately estimated and that some of the cell-based data were unsound. Among 69 samples of CLL cells, in which one copy of chromosome 13q was lost, we did not identify mutations within a 581-bp segment encompassing the miR-15a–16-1 gene. We also consider it very surprising that a single substitution outside the pri-miR-16-1 hairpin abolished the expression of both miR-15a and miR-16-1 as presented in Figure 2E of the original article. Figure 2E is inconclusive and mislabeled. It appears that miR-15a (mislabeled as U6, which is a 100-nucleotide small nuclear RNA) is present in all samples and 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 unexplained why miR-15a was detected in the left portion of Figure 2E but miR-16-1 was not detected in the right portion, showing the control human embryonic kidney 293 cells.

Arndt Borkhardt, M.D.
Uta Fuchs, Ph.D.
Ludwig-Maximilians-Universität München, 80337 Munich, Germany
arndt.borkhardt@med.uni-muenchen.de

Tom Tuschl, Ph.D.
Rockefeller University, New York, NY 10021

1 References

1. 1

   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
   Full Text | Web of Science | Medline

To the Editor:

Calin et al. found a significant association between a microRNA expression signature and ZAP-70 expression, mutational status of the immunoglobulin heavy-chain variable-region (IgV_H ) gene, and time to initial treatment in patients with CLL. They suggest that microRNA expression can be included as a prognostic marker in CLL. This proposal is premature, since the microRNA signature appears to be a surrogate for high ZAP-70 expression and unmutated IgV_H , which are known to be markers of a poor prognosis in patients with CLL.1-3 Was a multivariate analysis done to show that microRNA is an independent prognostic factor? Furthermore, it is not clear that the list of microRNA genes that predict time to treatment completely overlaps with the microRNAs that are associated with ZAP-70 expression and IgV_H mutational status (only five are the same), especially since the chip used to detect microRNA can discriminate between similar isoforms of microRNAs. Finally, it is not clear whether the set of nine microRNAs that predict progression was validated in the independent set of 50 patients with CLL.

Wee J. Chng, M.D.
National University Hospital, Singapore 119074
chngwj@nuh.com.sg

3 References

1. 1

   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
   Full Text | Web of Science | Medline

2. 2

   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
   Full Text | Web of Science | Medline

3. 3

   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
   Web of Science | Medline

Author/Editor Response

In our article, we reported the sequences of 42 microRNA genes in 75 patients with CLL and found mutations in 5 of the 42 genes in 11 patients. For this reason, we are surprised that Borkhardt et al. questioned our estimates on the basis of their failure to find mutations in the miR-15a–miR-16-1 transcript in 69 cases of CLL. That Borkhardt et al. did not find this rare germ-line mutation, which we identified in only 2 of 75 patients, is not unexpected. Moreover, the frequency of germ-line mutations may vary in different populations,1 and one of our patients with a germ-line mutation in miR-15a–miR-16-1 had familial CLL. The reduction in gene expression induced by this mutation was reproduced in two independent experiments and by a quantitative reverse-transcriptase–polymerase-chain-reaction assay. The mechanism responsible for this effect remains to be identified. Borkhardt et al. are correct that Figure 2E is mislabeled. The headings were reversed; the left portion should read “miR-16-1,” and the right portion should be labeled “miR-15a.”

Contrary to the assertion by Chng, our extended signature, associated with ZAP-70 expression (presented in the Supplementary Appendix to the article) and composed of 19 microRNAs, includes all 9 microRNAs that predict time to treatment (and not 5, as stated by Chng). We did not conclude that microRNA expression should substitute for ZAP-70 expression and IgV_H mutation status as a prognostic marker in cases of CLL but, rather, that it is a new prognostic marker. Whether it is an independent prognostic factor has yet to be determined. Finally, since we had no survival data for the test set of 50 samples of CLL, we restricted our analyses 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@osumc.edu

1 References

1. 1

   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
   CrossRef | Web of Science | Medline

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   Thorsten Zenz, Daniel Mertens, Hartmut Döhner, Stephan Stilgenbauer. (2008) Molecular diagnostics in chronic lymphocytic leukemia – Pathogenetic and clinical implications. Leukemia & Lymphoma 49:5, 864-873
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