Abstract 3290: Limited intra-tumor versus inter-tumor heterogeneity as assessed by proteomic profiling of high-risk neuroblastoma
Authors: Castellanos Raquel , Heaton-Johnson Katherine , Chung Jonathan , Nieves Edward , Fremed Michael , Master Stephen R. , Weiser Daniel 
Published: August 2015

Introduction: Tumor heterogeneity at a histological and molecular level has been previously described in neuroblastoma (NB) and therefore concern exists about how representative the molecular profile of a single tissue section can be of an entire tumor. We hypothesized that a single tissue section would provide a representative proteomic signature of the whole tumor.

Methods: As part of our effort to assess differential protein expression patterns from two maximally divergent groups with high-risk NB (i.e.: patients with early death from disease versus long-term survivors), we sampled multiple non-adjacent sections from blocks of 5 tumors within our larger cohort of 58 single section tumors. De-identified samples were obtained from the Children's Oncology Group biorepository. Using 5-10μ thick formalin fixed paraffin embedded tumor sections on a glass slide, we extracted 1 microgram of protein, performed tryptic digestion and desalting, and loaded peptides onto a reverse-phase column for separation by nanoflow chromatography. We performed peptide tandem mass spectrometry and protein expression vectors were identified using MaxQuant. The 1,495 most abundant proteins were evaluated across samples. Data were normalized and imputed. Hierarchical clustering analysis was performed using the pvclust R package between subjects with multiple sections and between all samples. We used average clustering criteria, correlation methods for distance, and pairwise covariance measures. Confidence of clusters was measured using approximately unbiased and bootstrap probability (1000 permutations) methods.

Results: Intra-tumor distance was significantly shorter than the inter-tumor distance. When testing only subjects with multiple tumor samples, 4 out of 5 samples from the same subject clustered with &gt;95% confidence. The samples from the remaining subject have a confidence &gt;80% but seem to have differing rates of missing data, suggesting experimental errors. When using all samples available, tumor samples from the same subject still clustered together and had a greater correlation than tumor samples from different subjects. A validation cohort of 7 distinct sections from separate blocks of tumors of 3 high-risk patients confirmed that tumors cluster by protein expression.

Conclusion and Future Directions: Intra-tumor heterogeneity exists but is significantly less than inter-tumor heterogeneity as assessed by proteomic profiling. We provide rationale to use single tissue section proteomics in ongoing research to define biologic drivers of primary refractory NB, a clinically important subgroup of patients who have highly lethal disease.

Citation Format: Raquel Castellanos, Katherine Heaton-Johnson, Jonathan Chung, Edward Nieves, Michael Fremed, Stephen R. Master, Daniel Weiser. Limited intra-tumor versus inter-tumor heterogeneity as assessed by proteomic profiling of high-risk neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3290. doi:10.1158/1538-7445.AM2015-3290