Tumor-Initiating Cells in Childhood Neuroblastoma—Response

We agree that the lines in question were contaminated by Epstein Barr Virus (EBV) and immediately withdrawn from circulation. In contrast to the note of caution expressed by Påhlman about using these 3 neuroblastoma tumor-initiating lines (NB12, NB88R2, and NB122R), we have recommended to the limited number of laboratories that were sent the lines or their derivatives that all stocks of them be destroyed. These lines were distributed under the condition that they are not to be circulated or provided to other laboratories, so we were confident that these lines no longer exist outside of our laboratory. Both Påhlman and we have noted neural characteristics in the lines, including widespread neural marker expression in tumor-initiating cells depleted of HIF-2α in xenografts (1) and neurosecretory granules by electron microscopy of NB88R2 xenografts (Fig. 1). Påhlman also published that downregulation of HIF-2α in the EBV-contaminated tumor-initiating cell lines resulted in a marked upregulation of neural and neuroblastoma-related gene expression in culture, indicating the persistence of neuroblastoma characteristics at later passages (1), at least in a subpopulation of the cells. Other neuroblastoma lines reported in the work of Hansford and colleagues (2) including NB04, NB05b, NB07, NB08, NB17, NB19, NB20, NB24, and NB32 were not contaminated with EBV as assessed by quantitative real-time PCR for EBNA1 (Table 1), whereas derivatives of NB12 as determined by short tandem repeat (STR) analysis including NB25, NB61, and NB67 expressed EBER1 (Table 1). NB88R2 and NB122R were first reported in the study by Pietras and colleagues, a publication from the Påhlman laboratory, with cell lines supplied by our laboratory (1).

Regarding the unpublished B-cell marker expression data in NB12, NB88R2, and NB122R that we reported at a meeting, we note that others have reported leukemia/lymphoma marker expression in neuroblastoma cell lines (3). The characterization of leukemia/lymphoma marker expression in non–EBV-contaminated neuroblastoma TIC lines is currently being examined and will be the subject of a future report.

We have reported that drugs that are effective on EBV-contaminated neuroblastoma TICs are effective on neuroblastoma established cell lines (4, 5) and we find that they are also effective on a non–EBV-contaminated primary neuroblastoma sphere-forming line (NB153). Our characterization of drug responses on NB153 and other neuroblastoma lines will also be the subject of a future report.

We thank Påhlman and colleagues for bringing this issue to our attention.

See the original Letter to the Editor, p. 821