New Molecular Signatures of High-Risk Neuroblastoma

Nai-Kong Cheung, MD, PhD

High-risk neuroblastoma cells harbor distinguishing molecular signatures, according to research that physician-scientists at Memorial Sloan Kettering Cancer Center and Weill Cornell Medicine published recently in the journal Neoplasia.

These unique characteristics distinguish neuroblastoma from other cancers. They could serve as useful biomarkers for high-risk neuroblastoma and may be potential targets for developing new treatments.

We analyzed a panel of five cell lines and 30 surgical samples of high-risk neuroblastoma tumors using assays that assess telomere length, length distribution patterns, single-stranded DNA on the G and C strands, and extrachromosomal circular telomeres. (1)

We were surprised to find high levels of C-strand single-strand DNA overhangs and t-circles, consistent with active telomere trimming. These unique characteristics distinguish neuroblastoma from other cancers. They could serve as useful biomarkers for high-risk neuroblastoma and may be potential targets for developing new treatments. (1)

High-Risk Neuroblastoma and Telomeres

Neuroblastoma develops in the sympathetic nervous system, forming tumors in the adrenal glands, abdomen, neck, chest, or pelvis. The tumors can also spread to other parts of the body, such as the bone marrow and bones. Neuroblastoma accounts for 6 percent of all pediatric tumors but more than 10 percent of mortality, making it one of the most aggressive and lethal childhood cancers. (2) There are about 800 new diagnoses of neuroblastoma each year in the United States. (3)

Telomeres are repeated sequences of DNA positioned at the end of chromosomes. They play essential roles in genome stability and controlling cell proliferation. An adequate amount of telomeric DNA is necessary to sustain cell proliferation. A common hallmark of cancer cells is their ability to upregulate telomere maintenance mechanisms, enabling the cells to keep replicating. (4)

Genomic studies of high-risk neuroblastoma have identified MYCN, TERT, and ATRX mutations as frequent and mutually exclusive drivers of the disease. (5) Telomere DNA is most often maintained by telomerase activation, which is strongly associated with TERT mutations and MYCN amplifications. (6), (7) However, up to 25 percent of neuroblastoma tumors replenish telomere DNA through the alternative lengthening of telomeres (ALT) pathway, which is linked to ATRX mutations and deletions. (8), (9)

There is a growing body of evidence suggesting that telomere-specific characteristics — length, heterogeneity, and extrachromosomal telomeric DNA — are independent prognostic indicators of high-risk disease. (8), (10), (11) This evidence implies that telomeres and telomere maintenance mechanisms are biologically and clinically relevant to the underlying pathogenesis of neuroblastoma. Further, a recent extensive genetic profile of neuroblastoma tumors strongly suggested that telomere maintenance mechanisms are an important prognostic indicator. (12)

Previous studies have characterized telomere structures in neuroblastoma by analyzing average telomere lengths by Southern analysis and C-circle levels as markers of ALT activity. But other significant telomere features may also play a role in tumor development. For example, levels of single-stranded DNA on either the G or C strands could indicate abnormal telomere metabolism. Normal telomeres usually have a short (50 to 200 nt) 3’-overhang of the G strand, but “deprotected” telomeres can carry much longer overhangs due to degradation of the C strand. (13) Other research has reported 5’-overhangs of the C strand, suggesting that the feature may be a marker of telomere recombination and ALT. (14), (15), (16) Additionally, rather than average telomere length, the fraction of short telomeres in a cell may be a better measure of cell proliferation, since only a few abnormally short telomeres are sufficient to trigger cell death. (17)

Study Design

Our study was the first to investigate the single-stranded G and C overhangs of telomeres in multiple cell lines and tumor samples of high-risk neuroblastoma. We applied a series of assays to examine different aspects of telomere structures, making modifications to several existing assays to reveal more features, and also used the STELA assay to assess the levels of short telomeres. (18), (19), (20) STELA has typically been used to analyze primary cells, such as fibroblasts and lymphocytes, (19), (20), (21) not for the comprehensive profiling of human cancers.

We used archived neuroblastoma tumor samples from 30 patients with metastatic disease who were treated at MSK. Three of the neuroblastoma cell lines were created at MSK, and two were provided by Robert Seeger of Children’s Hospital Los Angeles. To compare our results with other cancers, we used primary acute myeloid leukemia specimens provided by Weill Cornell Medicine and leukemia cell lines obtained from the American Type Culture Collection (ATCC) or the German repository DSMZ, as well as HeLa cells and the osteosarcoma cell line U2OS, also from ATCC. All human tissue samples were obtained with informed consent and Institutional Review Board approvals at their respective organizations.

Study Findings

We were surprised to find several previously undiscovered characteristics of high-risk neuroblastoma. Unlike other cancers, we found consistently high levels of C-strand overhangs and t-circles that were consistent with active telomere trimming. These features were observed in both telomerase and ALT tumors, regardless of telomere length distribution. (1)

By analyzing telomere length distribution via STELA, we observed variable and poor amplification of telomere DNA, indicating substantial telomere damage. Further analysis determined that this damage included 8-oxo-2’-deoxyguanine and other lesions. Notably, we observed telomere trimming in normal neural tissues, suggesting that telomere maintenance mechanisms in high-risk neuroblastoma may evolve in a canonical setting of telomere shortening. (1)

Together, our findings suggest that telomere trimming by itself may distinguish neuroectodermal-derived tumors from other human cancers. These unique features have both biologic and therapeutic implications. To understand the origins of these unique features, the collaborating teams (the Cheung lab at MSK and the Lue lab at Weill Cornell Medicine) are expanding their analysis to more tumor samples and investigating the protein compositions of telomeres.

More work lies ahead to fully exploit these discoveries in cancer diagnosis and therapies.

Advancing Pediatric Neuroblastoma Care

At MSK Kids, our neuroblastoma team of experts is dedicated to improving survival rates and the quality of life of children with neuroblastoma. We treat more children with neuroblastoma than any other cancer center in the world. Today, more than 50 percent of the patients treated for neuroblastoma at MSK survive, compared to less than 5 percent in the 1980s. Localized neuroblastoma is highly curable today; many patients can be treated effectively with surgery alone.

To complement the neuroblastoma clinical program at MSK, the Cheung lab focuses on engineering antibodies and immune cells to treat both solid tumors and liquid tumors in children. By integrating multimodality strategies — including surgery, chemotherapy, radiation therapy, isotretinoin, and immunotherapy — the survival rates for patients with high-risk metastatic neuroblastoma have substantially improved. We continue to adopt new drugs to overcome the resistance that neuroblastoma often develops after prolonged genotoxic therapies.

Currently 19 clinical trials for neuroblastoma are active at MSK, testing biologic agents, antibodies, vaccines, natural killer cells, radioisotopes, and combinations of novel therapies with conventional treatments. MSK continues to investigate the underlying biology of neuroblastoma to look for its vulnerabilities in the new era of precision medicine.

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This telomere work was supported by Dr. Cheung’s endowed Enid A. Haupt Chair in Pediatric Oncology, the Robert Steel Foundation, and the Catie Hoch Foundation. Dr. Cheung’s conflicts of interest are detailed here. Refer to the paper in Neoplasia regarding the supports for this study provided to authors at the collaborating institutions.


  1. Yu EY, Cheung IY, Feng Y, et al. Telomere Trimming and DNA Damage as Signatures of High Risk Neuroblastoma [Epub ahead of print, 2019 May 23]. Neoplasia. 2019;21(7):689–701. 
  2. Ward E, DeSantis C, Robbins A et al. Childhood and adolescent cancer statistics, 2014. CA Cancer J Clin. 2014;64: 83–103.
  3. Key Statistics About Neuroblastoma. American Cancer Society. Accessed on June 11, 2019. 
  4. Hanahan D and Weinberg RA (2011). Hallmarks of cancer: the next generation. Cell. 2011;114:646–674.
  5. Hertwig F, Peifer M, and FischerM. Telomere maintenance is pivotal for high-risk neuroblastoma. Cell Cycle. 2016;15,311–312.
  6. Hiyama E, Hiyama K, Yokoyama T, et al. Correlating telomerase activity levels with human neuroblastoma outcomes. Nat Med. 1995;1:249–255.
  7. Peifer M, Hertwig F, Roels F, et al. Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Nature. 2015;526:700–704.
  8. Cheung NK, Zhang J, Lu C, et al. Association of age at diagnosis and genetic mutations in patients with neuroblastoma. JAMA. 2012;307(10):1062–1071.
  9. Dagg RA, Pickett HA, Neumann AA, et al. Extensive Proliferation of Human Cancer Cells with Ever-Shorter Telomeres. Cell Rep. 2017;19:2544–2556.
  10. Farooqi AS, Dagg RA, Choi LM, et al. Alternative lengthening of telomeres in neuroblastoma cell lines is associated with a lack of MYCN genomic amplification and with p53 pathway aberrations. J Neurooncol. 2014;119:17–26.
  11. Ohali A, Avigad S, Ash S, et al. Telomere length is a prognostic factor in neuroblastoma. Cancer. 2006;107:1391–1399.
  12. Ackermann S, Cartolano M, Hero B, et al. A mechanistic classification of clinical phenotypes in neuroblastoma. Science. 2018;362:1165–1170.
  13. de Lange T (2018). Shelterin-Mediated Telomere Protection. Annu Rev Genet. 2018;52:223–247.
  14. Oganesian L and Karlseder J. Mammalian 5’ C-rich telomeric overhangs are a mark of recombination-dependent telomere maintenance. Mol Cell. 2011;42:224–236.
  15. Oganesian L and Karlseder J. 5’ C-rich telomeric overhangs are an outcome of rapid telomere truncation events. DNA Repair. 2013;12:,238–245.
  16. Rivera T, Haggblom C, Cosconati S, and Karlseder J. A balance between elongation and trimming regulates telomere stability in stem cells. Nat Struct Mol. Biol. 2017;24,30–39.
  17. Kaul Z, Cesare AJ, Huschtscha LI, et al. Five dysfunctional telomeres predict onset of senescence in human cells. EMBO Rep. 2011;13:52–59.
  18. Baird DM, Rowson J, Wynford-Thomas D, and Kipling D. Extensive allelic variation and ultrashort telomeres in senescent human cells. Nat Genet. 2003;33:203–207.
  19. Bendix L, Horn PB, Jensen UB, et al. The load of short telomeres, estimated by a new method, Universal STELA, correlates with number of senescent cells. Aging Cell. 2010;9:383–397.
  20. Lai TP, Zhang N, Noh J, et al. A method for measuring the distribution of the shortest telomeres in cells and tissues. Nat Commun. 2017;8:1356.
  21. Huang EE, Tedone E, O’Hara R, et al. The maintenance of telomere length in CD28+ T cells during T lymphocyte stimulation. Sci Rep. 2017;7(1):6785.