Molecular signature incorporating the immune microenvironment enhances thyroid cancer outcome prediction.

Genomic and transcriptomic analysis has furthered our understanding of many tumors. Yet, thyroid cancer management is largely guided by staging and histology, with few molecular prognostic and treatment biomarkers. Here, we utilize a large cohort of 251 patients with 312 samples from two tertiary medical centers and perform DNA/RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to identify biomarkers of aggressive thyroid malignancy. We identify high-risk mutations and discover a unique molecular signature of aggressive disease, the Molecular Aggression and Prediction (MAP) score, which provides improved prognostication over high-risk mutations alone. The MAP score is enriched for genes involved in epithelial de-differentiation, cellular division, and the tumor microenvironment. The MAP score also identifies aggressive tumors with lymphocyte-rich stroma that may benefit from immunotherapy. Future clinical profiling of the stromal microenvironment of thyroid cancer could improve prognostication, inform immunotherapy, and support development of novel therapeutics for thyroid cancer and other stroma-rich tumors.

Identifying the effectiveness of 3D culture systems to recapitulate breast tumor tissue in situ.

PURPOSE: Breast cancer heterogeneity contributes to chemotherapy resistance and decreased patient survival. To improve patient outcomes it is essential to develop a technology that is able to rapidly select the most efficacious therapy that targets the diverse phenotypes present within the tumor. Breast cancer organoid technologies are proposed as an attractive approach for evaluating drug responses prior to patient therapy. However, there remain challenges in evaluating the effectiveness of organoid cultures to recapitulate the heterogeneity present in the patient tumor in situ. METHOD: Organoids were generated from seven normal breast and nineteen breast cancer tissues diagnosed as estrogen receptor positive or triple negative. The Jensen-Shannon divergence index, a measure of the similarity between distributions, was used to compare and evaluate heterogeneity in starting tissue and their resultant organoids. Heterogeneity was analyzed using cytokeratin 8 and cytokeratin 14, which provided an easily scored readout. RESULTS: In the in vitro culture system HER1 and FGFR were able to drive intra-tumor heterogeneity to generate divergent phenotypes that have different sensitivities to chemotherapies. CONCLUSION: Our methodology, which focuses on quantifiable cellular phenotypes, provides a tractable system that complements omics approaches to provide an unprecedented view of heterogeneity and will enhance the identification of novel therapies and facilitate personalized medicine.

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes.

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

Proline rich 11 (PRR11) overexpression amplifies PI3K signaling and promotes antiestrogen resistance in breast cancer.

The 17q23 amplicon is associated with poor outcome in ER+ breast cancers, but the causal genes to endocrine resistance in this amplicon are unclear. Here, we interrogate transcriptome data from primary breast tumors and find that among genes in 17q23, PRR11 is a key gene associated with a poor response to therapeutic estrogen suppression. PRR11 promotes estrogen-independent proliferation and confers endocrine resistance in ER+ breast cancers. Mechanistically, the proline-rich motif-mediated interaction of PRR11 with the p85α regulatory subunit of PI3K suppresses p85 homodimerization, thus enhancing insulin-stimulated binding of p110-p85α heterodimers to IRS1 and activation of PI3K. PRR11-amplified breast cancer cells rely on PIK3CA and are highly sensitive to PI3K inhibitors, suggesting that PRR11 amplification confers PI3K dependence. Finally, genetic and pharmacological inhibition of PI3K suppresses PRR11-mediated, estrogen-independent growth. These data suggest ER+/PRR11-amplified breast cancers as a novel subgroup of tumors that may benefit from treatment with PI3K inhibitors and antiestrogens.

RSK2 Maintains Adult Estrogen Homeostasis by Inhibiting ERK1/2-Mediated Degradation of Estrogen Receptor Alpha.

In response to estrogens, estrogen receptor alpha (ERα), a critical regulator of homeostasis, is degraded through the 26S proteasome. However, despite the continued presence of estrogen before menopause, ERα protein levels are maintained. We discovered that ERK1/2-RSK2 activity oscillates during the estrous cycle. In response to high estrogen levels, ERK1/2 is activated and phosphorylates ERα to drive ERα degradation and estrogen-responsive gene expression. Reduction of estrogen levels results in ERK1/2 deactivation. RSK2 maintains redox homeostasis, which prevents sustained ERK1/2 activation. In juveniles, ERK1/2-RSK2 activity is not required. Mammary gland regeneration demonstrates that ERK1/2-RSK2 regulation of ERα is intrinsic to the epithelium. Reduced RSK2 and enrichment in an estrogen-regulated gene signature occur in individuals taking oral contraceptives. RSK2 loss enhances DNA damage, which may account for the elevated breast cancer risk with the use of exogenous estrogens. These findings implicate RSK2 as a critical component for the preservation of estrogen homeostasis.

ER+ Breast Cancers Resistant to Prolonged Neoadjuvant Letrozole Exhibit an E2F4 Transcriptional Program Sensitive to CDK4/6 Inhibitors.

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole.Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months).Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E-15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC.Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients' ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517-29. ©2018 AACR.

Alternative splicing of ALCAM enables tunable regulation of cell-cell adhesion through differential proteolysis.

While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.

Genotypic and clinical heterogeneity within NCCN favorable-risk acute myeloid leukemia.

The National Comprehensive Cancer Network (NCCN) defines the following types of acute myeloid leukemia (AML) as favorable-risk: acute promyelocytic leukemia with t(15;17) (APL); AML with core-binding factor (CBF) rearrangements, including t(8;21) and inv(16) or t(16;16) without mutations in KIT (CBF-KITwt); and AML with normal cytogenetics and mutations in NPM1 (NPM1mut); or biallelic mutations in CEBPA (CEBPAmut/mut), without FLT3-ITD. Although these AMLs are categorized as favorable risk by NCCN, clinical experience suggests that there are differences in clinical outcome amongst these cytogenetically and molecularly distinct leukemias. This study compared clinical and genotypic characteristics of 60 patients with favorable-risk AML, excluding APL, and demonstrated significant differences between them. Patients with NPM1mut AML were significantly older than those in the other groups. Targeted next-generation sequencing on DNA from peripheral blood or bone marrow revealed significantly more mutations in NPM1mut AML than the other favorable-risk diseases, especially in genes related to DNA splicing and methylation. CEBPAmut/mut AMLs exhibited more mutations in transcription-related genes. Patients with NPM1mut AML and CEBPAmut/mut AML show significantly reduced overall survival in comparison with CBF-KITwt AML. These findings emphasize that favorable-risk AML patients have divergent outcomes and that differences in clinical and genotypic characteristics should be considered in their evaluation and management.

Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance.

Inhibition of proliferation in estrogen receptor-positive (ER+) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER+/human epidermal growth factor receptor 2-negative (HER2-) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including FGFR1 and CCND1, respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER+ tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER+FGFR1/CCND1 coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal ESR1 fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.

Extracellular Matrix/Integrin Signaling Promotes Resistance to Combined Inhibition of HER2 and PI3K in HER2+ Breast Cancer.

PIK3CA mutations are associated with resistance to HER2-targeted therapies. We previously showed that HER2+/PIK3CAH1047R transgenic mammary tumors are resistant to the HER2 antibodies trastuzumab and pertuzumab but respond to PI3K inhibitor buparlisib (TPB). In this study, we identified mechanisms of resistance to combined inhibition of HER2 and PI3K. TPB-resistant tumors were generated by treating HER2+/PIK3CAH1047R tumor-bearing mice long term with the drug combination. RNA sequencing of TPB-resistant tumors revealed that extracellular matrix and cell adhesion genes, including collagen II (Col2a1), were markedly upregulated, accompanied by activation of integrin ß1/Src. Cells derived from drug-resistant tumors were sensitive to TBP when grown in vitro, but exhibited resistance when plated on collagen or when reintroduced into mice. Drug resistance was partially reversed by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoate. Inhibition of integrin ß1/Src blocked collagen-induced resistance to TPB and inhibited growth of drug-resistant tumors. High collagen II expression was associated with significantly lower clinical response to neoadjuvant anti-HER2 therapy in HER2+ breast cancer patients. Overall, these data suggest that upregulation of collagen/integrin/Src signaling contributes to resistance to combinatorial HER2 and PI3K inhibition. Cancer Res; 77(12); 3280-92. ©2017 AACR.

Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+) subtype and fourteen triple negative (TN) subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

Diverse, Biologically Relevant, and Targetable Gene Rearrangements in Triple-Negative Breast Cancer and Other Malignancies.

Triple-negative breast cancer (TNBC) and other molecularly heterogeneous malignancies present a significant clinical challenge due to a lack of high-frequency "driver" alterations amenable to therapeutic intervention. These cancers often exhibit genomic instability, resulting in chromosomal rearrangements that affect the structure and expression of protein-coding genes. However, identification of these rearrangements remains technically challenging. Using a newly developed approach that quantitatively predicts gene rearrangements in tumor-derived genetic material, we identified and characterized a novel oncogenic fusion involving the MER proto-oncogene tyrosine kinase (MERTK) and discovered a clinical occurrence and cell line model of the targetable FGFR3-TACC3 fusion in TNBC. Expanding our analysis to other malignancies, we identified a diverse array of novel and known hybrid transcripts, including rearrangements between noncoding regions and clinically relevant genes such as ALK, CSF1R, and CD274/PD-L1 The over 1,000 genetic alterations we identified highlight the importance of considering noncoding gene rearrangement partners, and the targetable gene fusions identified in TNBC demonstrate the need to advance gene fusion detection for molecularly heterogeneous cancers. Cancer Res; 76(16); 4850-60. ©2016 AACR.

Aberrant activation of NF-κB signaling in mammary epithelium leads to abnormal growth and ductal carcinoma in situ.

BACKGROUND: Approximately 1 in 5 women diagnosed with breast cancer are considered to have in situ disease, most often termed ductal carcinoma in situ (DCIS). Though recognized as a risk factor for the development of more invasive cancer, it remains unclear what factors contribute to DCIS development. It has been shown that inflammation contributes to the progression of a variety of tumor types, and nuclear factor kappa B (NF-κB) is recognized as a master-regulator of inflammatory signaling. However, the contributions of NF-κB signaling to tumor initiation are less well understood. Aberrant up-regulation of NF-κB activity, either systemically or locally within the breast, could occur due to a variety of commonly experienced stimuli such as acute infection, obesity, or psychological stress. In this study, we seek to determine if activation of NF-κB in mammary epithelium could play a role in the formation of hyperplastic ductal lesions. METHODS: Our studies utilize a doxycycline-inducible transgenic mouse model in which constitutively active IKKß is expressed specifically in mammary epithelium. All previously published models of NF-κB modulation in the virgin mammary gland have been constitutive models, with transgene or knock-out present throughout the life and development of the animal. For the first time, we will induce activation at later time points after normal ducts have formed, thus being able to determine if NF-κB activation can promote pre-malignant changes in previously normal mammary epithelium. RESULTS: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures. Short-term activation created hyperproliferative, enlarged ducts with filled lumens. Increased expression of inflammatory markers was concurrent with the down-regulation of hormone receptors and markers of epithelial differentiation. Furthermore, the oncoprotein mucin 1, known to be up-regulated in human and mouse DCIS, was over-expressed and mislocalized in the activated ductal tissue. CONCLUSIONS: These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer. Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

Integrative analysis of head and neck cancer identifies two biologically distinct HPV and three non-HPV subtypes.

PURPOSE: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment. EXPERIMENTAL DESIGN: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV(+) tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics. RESULTS: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV(+) and one HPV(-) subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8(+) lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes. CONCLUSION: Our five-subtype classification provides a comprehensive overview of HPV(+) as well as HPV(-) HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC.

Surgical resection margins in desmoid-type fibromatosis: a critical reassessment.

The prognostic value of surgical resection margins in predicting recurrence of desmoid-type fibromatosis remains inconclusive, and appropriate resection margins are not defined. We analyzed whether specific factors related to margin status, including the adequacy of pathologic assessment, affect the prognostic impact of surgical resection margins in a retrospective cohort study. The distance (clearance) and tissue composition of resection margins were recorded for primary desmoid resections with negative margins. The number of positive margins and the linear extent of marginal involvement were recorded for cases with positive margins. The number of tissue sections submitted for histopathologic evaluation of resection margins was also noted. Factors predictive of a positive margin and local recurrence were evaluated by logistic regression and survival analyses. The probability of documenting a positive margin increased with the number of tissue sections submitted for assessment of margin status (odds ratio 1.16; P=0.007). Recurrence-free survival at 10 years for patients with positive (N=52) or negative (N=40) resection margins was 68% and 60%, respectively. Only tumor clearance was predictive of local recurrence. A positive or close (<1 mm) resection margin was an independent prognostic factor for local recurrence in a multivariate Cox regression model accounting for patient age and anatomic site (hazard ratio 9.52; P=0.028). Surgical resection margin status, as currently evaluated and reported, fails to predict recurrence-free survival for desmoid tumors. Instead, positive or close (<1 mm) resection margins are predictive of disease recurrence. In addition, multiple targeted sections are necessary for accurate assessment of surgical resection margins of desmoid-type fibromatosis.

Detection of internal exon deletion with exon Del.

BACKGROUND: Exome sequencing allows researchers to study the human genome in unprecedented detail. Among the many types of variants detectable through exome sequencing, one of the most over looked types of mutation is internal deletion of exons. Internal exon deletions are the absence of consecutive exons in a gene. Such deletions have potentially significant biological meaning, and they are often too short to be considered copy number variation. Therefore, to the need for efficient detection of such deletions using exome sequencing data exists. RESULTS: We present ExonDel, a tool specially designed to detect homozygous exon deletions efficiently. We tested ExonDel on exome sequencing data generated from 16 breast cancer cell lines and identified both novel and known IEDs. Subsequently, we verified our findings using RNAseq and PCR technologies. Further comparisons with multiple sequencing-based CNV tools showed that ExonDel is capable of detecting unique IEDs not found by other CNV tools. CONCLUSIONS: ExonDel is an efficient way to screen for novel and known IEDs using exome sequencing data. ExonDel and its source code can be downloaded freely at https://github.com/slzhao/ExonDel.

Desmoid-type fibromatosis-associated Gardner fibromas: prevalence and impact on local recurrence.

Although Gardner fibroma is a precursor lesion of desmoid tumor, the prevalence and prognostic importance of Gardner fibroma associated with desmoid tumors has not been systematically studied in adults. From 129 patients with desmoid-type fibromatosis, 170 specimens were re-examined for the presence of an associated Gardner fibroma. Clinicopathologic features of Gardner fibroma-associated desmoid-type fibromatosis were compared to desmoid tumors without associated Gardner fibroma. Recurrence-free survival was compared using multivariate Cox proportional hazard regression to account for known confounding factors. Of 104 evaluable primary desmoid tumor resections, 25 (24%) had an associated Gardner fibroma. When previous incisional biopsies and resection specimens of locally recurrent desmoid tumors were also examined, the overall prevalence of associated Gardner fibroma was 37%. Desmoid tumors arising in high risk anatomic sites (extremities or deep soft tissues of the back and chest wall) were more often associated with Gardner fibroma than tumors at other sites. Median recurrence-free survival for patients with Gardner fibroma-associated desmoid-type fibromatosis was 3.2 years, whereas median survival for patients without associated Gardner fibroma was >25 years (hazard ratio 2.8; P = 0.001). Although the presence of Gardner fibroma had no impact on the recurrence rate of desmoid tumors arising at high risk anatomic sites, associated Gardner fibroma increased the risk of recurrence 4-fold for desmoid tumors at low risk anatomic sites. Associated Gardner fibroma is under-recognized in desmoid-type fibromatosis and increases the risk of local recurrence for a subgroup of patients.

Validation of a prognostic multi-gene signature in high-risk neuroblastoma using the high throughput digital NanoString nCounter™ system.

Microarray-based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high-quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin-fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high-throughput digital system to assay the expression of genes in an "ultra-high risk" microarray classifier in FFPE high-risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi-gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high-risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearson's correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42-gene panel sub-stratified the high-risk cohort into two subsets with statistically significantly different overall survival (p = 0.0027) and event-free survival (p = 0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high-risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high-risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.