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Supplementary Table 1 XLS file 22K, Tumors and reference tissues for methylation microarray analysis

Supplementary Figure 1 - PDF file 1233K, Supplemental Figure 1: Representative array comparative genomic hybridization (aCGH) results of genomic copy number near-normal GIST (left column, n=5) and genomic copy number abnormal GIST (right column, n=5) (Agilent 180K catalogue assay)

Supplementary Table 2 XLSX file 490K, Illumina GoldenGate Methylation Standard Cancer Panel I Target CpG Annotations and differential methylation significance values for group comparisons listed in Table

Legends for all included Supplementary Figures

Heat map of cell cycle gene expression. Gene expression was normalized and compared between the primary MTC, metastasis one, and metastasis two lesions from the whole transcriptome RNA-seq data. Purple indicates lower and yellow indicates higher expression. Values to generate colors are z-scores across each genes' relative transcript levels [log2(FPKM)].

Weight-for-age and height-for-age growth curves. Male patient weights (A), heights (B) and female patient weights (C), and heights (D) were monitored during the follow up period. Patient weight-for-age and height-for-age values were compared to U.S. Center for Disease Control reference growth curves.

Doubling time of calcitonin and CEA for patient #12 was suggestive of progressive disease. Patient #12 experienced best response 434 days after starting vandetanib, with stable lesions in the thyroid bed (showed on T2-weighted MRI of the neck, A) and the mediastinum (showed on CT of the chest, B). In August 2016, the sum of longest diameter (per RECIST) of target lesions was consistent with SD (C,D) (note that not all target lesions are shown). Biomarker response in the two years prior (E) showed CEA doubling time 1.2 years (F) and calcitonin doubling time 1.67 years (G). Patient #12 was confirmed to have PD via RECIST February 2017.

Purpose: Vandetanib is well-tolerated in patients with advanced medullary thyroid carcinoma (MTC). Long-term outcomes and mechanisms of MTC progression have not been reported previously.Experimental Design: We monitored toxicities and disease status in patients taking vandetanib for hereditary, advanced MTC. Tumor samples were analyzed for molecular mechanisms of disease progression.Results: Seventeen patients [8 male, age 13 (9–17)* years] enrolled; 16 had a RET p.Met918Thr germline mutation. The duration of vandetanib therapy was 6.1 (0.1–9.7+)* years with treatment ongoing in 9 patients. Best response was partial response in 10, stable disease in 6, and progressive disease in one patient. Duration of response was 7.4 (0.6–8.7+)* and 4.9 (0.6–7.8+)* years in patients with PR and SD, respectively. Six patients died 2.0 (0.4–5.7)* years after progression. Median progression-free survival (PFS) was 6.7 years [95% confidence interval (CI): 2.3 years–undefined] and 5-year overall survival (OS) was 88.2% (95% CI: 60.6%–96.9%). Of 16 patients with a RET p.Met918Thr mutation, progression-free survival was 6.7 years (95% CI: 3.1–undefined) and 5-year overall survival was 93.8% (95% CI: 63.2%–99.1%). No patients terminated treatment because of toxicity. DNA sequencing of tissue samples (n = 11) identified an increase in copy number alterations across the genome as a potential mechanism of drug resistance [*median (range)].Conclusions: This study demonstrates that vandetanib is safe and results in sustained responses in children and adolescents with hereditary MTC. Our preliminary molecular data suggest that an increase in copy number abnormalities may be associated with tumor progression in hereditary MTC patients treated with vandetanib. Clin Cancer Res; 24(4); 753–65. ©2017 AACR.

Legends for all included Supplementary Figures

Doubling time of calcitonin and CEA during vandetanib therapy. Calcitonin and CEA slopes and doubling times were calculated from the change during the initial four months of vandetanib (A,B), from values the year before experiencing PD or the data cutoff (C,D) and in patients that experienced progressive disease (E).

Patients #2, #5, #10, #11, #13, #15, and #16 experienced durable SD or PR during the follow up period. Calcitonin and CEA are shown as percent change from baseline at the start of vandetanib therapy (left y-axis). Tumor size is the sum of longest diameter of lesions per RECISTv1.0 (right y-axis).

Osteosarcoma (OS) is the most common malignancy of the bone, typically diagnosed during the adolescent years. Despite early advances in treatment, OS survival rates have not improved in recent times. Targeted treatment remains elusive because OS is a genetically complex and heterogeneous disease and the low incidence of sporadic OS presents a challenge in dissecting the causes and drivers of tumor development. Type II Rothmund-Thomson Syndrome (RTS), a cancer predisposition disorder, presents a potential model for studying OS pathogenesis as roughly 30% of Type II RTS patients are eventually diagnosed with OS. Type II RTS is associated with germline mutations of the DNA helicase RECQL4 that exclusively target the C-terminal helicase domain thought to be involved in various cellular mechanisms of DNA repair. The N-terminal SLD2-homology domain, required for the assembly of the CMG complex at origins of replication, is spared. We used CRISPR/Cas9 gene editing to generate cell lines containing a common RECQL4 mutation identified in Type II RTS patients to further study its effects. Unexpectedly, a mutation previously reported as a nonsense mutation instead upregulates the activation of a known cryptic splice site in exon 14. This splice variant results in the in-frame deletion of the final 66 amino acids of exon 14. Our cell lines exhibit a mildly increased sensitivity to the DNA double-strand break inducing agent neocarzinostatin consistent with RECQL4’s reported functions in DNA damage repair. We will use next generation sequencing techniques to further characterize DNA damage repair signatures in RTS RECQL4 mutant cell lines. Citation Format: Tianyi Wu, Robert L. Walker, Marbin Pineda, MiYoung Lee, Yuelin Zhu, Ashok R. Venkitaraman, Paul S. Meltzer. Functional characterization of a RECQL4 mutation in Rothmund Thomson Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2559.

Background: Unlike recurrences with other therapies, ER+ breast cancers (BC) can recur ≥10 yrs after an apparent successful period of adjuvant endocrine therapy. The molecular basis for this pattern of resistance is unresolved. We addressed the hypothesis that early recurrences during tamoxifen (TAM) treatment exhibit different biological characteristics than those that recur years later by assessing for variation in their respective transcriptomes. Methods: Appropriate tumors were identified from a set (BC030280) of snap-frozen tumor biopsies collected in Edinburgh from subjects with stage I-III BC before starting TAM (no chemotherapy); all had ≥10 yrs follow-up. Using rigorous standard operating procedures, high quality total RNA was extracted from samples with ≥50% malignant epithelium and arrayed on Affymetrix U133 Plus 2.0 GeneChips. Raw data were normalized using PLIER and analyzed with an adapted validated training and internal cross-validation workflow to avoid gene selection bias. A published dataset (Loi) was used for independent classifier validation that best fit our criteria for sample size, treatment (TAM only), data quality, and recurrence distribution. Early (E) vs late (L) recurrences were defined as distant relapse ≤3 vs ≥10 yrs from diagnosis. To explore putative mechanistic associations driving the transcriptome differences, a novel computational procedure was developed to integrate gene expression data with protein-protein interaction (PPI) data and create a statistical network model of the signaling. Metropolis sampling, a Markov Chain Monte Carlo method that can be implemented as a modified random walk procedure, identified ER network topology represented by the genes (nodes) and their predicted interconnections (edges). Results: A support vector machine with recursive feature elimination was used for the binary classification tasks on the BC030280 dataset. The optimized classifier for E vs L recurrence was independently tested on the Loi dataset with high levels of accuracy, specificity, and sensitivity. Classifier validation is supported by the respective survival curves (not shown). Gene set enrichment analysis reveals the top PPIs are primarily related to apoptosis (23/50; p=2.9e-13) and proliferation (14/50; p=6.8e-5). Substantial overlap of the network features and topology was seen between datasets. Specifically, increased relative expression of ESR1, ESR2, EGFR, BCL2, and AR was seen in L vs E recurrent tumors, and increased expression of CALM1, CALM2, CALM3, SRC, CDK1, and MAPK1 was seen in E vs L recurrent tumors. Several hubs (nodes with ≥5 edges) independently predicted for recurrence in additional public datasets of ER+ BC. Discussion: Our work provides clear evidence that robust molecular differences exist between ER+ BC that recur early vs. much later despite adjuvant TAM. Exploiting these differences will improve our understanding of involved signaling pathways, allow for the reliable prediction of early treatment failure, and guide use of novel therapeutics specifically directed at preventing E vs L recurrences on endocrine therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-8.

In rats, prepubertal exposure to low-fat diet containing n-3 polyunsaturated fatty acids (PUFA) reduces mammary cell proliferation, increases apoptosis, and lowers risk of mammary tumors in adulthood, whereas prepubertal high-fat n-3 PUFA exposure has opposite effects. To identify signaling pathways mediating these effects, we performed gene microarray analyses and determined protein levels of genes related to mammary epithelial cell proliferation. Nursing female rats and rat pups were fed low-fat (16% energy from fat) or high-fat (39% energy from fat) n-3 or n-6 PUFA diets between postnatal days 5 and 24. cDNA gene expression microarrays were used to identify global changes in the mammary glands of 50-day-old rats. Differences in gene expression were confirmed by real-time quantitative PCR, and immunohistochemistry was used to assess changes in the peroxisome proliferator–activated receptor γ and cyclin D1 levels. DNA damage was determined by 8-hydroxy-2′-deoxyguanosine assay. Expressions of the antioxidant genes thioredoxin, heme oxygenase, NADP-dependent isocitrate dehydrogenase, and metallothionein III, as well as peroxisome proliferator–activated receptor γ protein, were increased in the mammary glands of 50-day-old rats prepubertally fed the low-fat n-3 PUFA diet. Prepubertal exposure to the high-fat n-3 PUFA diet increased DNA damage and cyclin D1 protein and reduced expression of BRCA1 and cardiotrophin-1. Reduction in mammary tumorigenesis among rats prepubertally fed a low-fat n-3 PUFA diet was associated with an up-regulation of antioxidant genes, whereas the increase in mammary tumorigenesis in the high-fat n-3 PUFA fed rats was linked to up-regulation of genes that induce cell proliferation and down-regulation of genes that repair DNA damage and induce apoptosis.

One-third of all estrogen receptor (ER)–positive breast tumors treated with endocrine therapy fail to respond, and the remainder is likely to relapse in the future. Almost all data on endocrine resistance has been obtained in models of invasive ductal carcinoma (IDC). However, invasive lobular carcinomas (ILC) comprise up to 15% of newly diagnosed invasive breast cancers each year and, whereas the incidence of IDC has remained relatively constant during the last 20 years, the prevalence of ILC continues to increase among postmenopausal women. We report a new model of Tamoxifen (TAM)-resistant invasive lobular breast carcinoma cells that provides novel insights into the molecular mechanisms of endocrine resistance. SUM44 cells express ER and are sensitive to the growth inhibitory effects of antiestrogens. Selection for resistance to 4-hydroxytamoxifen led to the development of the SUM44/LCCTam cell line, which exhibits decreased expression of ERα and increased expression of the estrogen-related receptor γ (ERRγ). Knockdown of ERRγ in SUM44/LCCTam cells by siRNA restores TAM sensitivity, and overexpression of ERRγ blocks the growth-inhibitory effects of TAM in SUM44 and MDA-MB-134 VI lobular breast cancer cells. ERRγ-driven transcription is also increased in SUM44/LCCTam, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity. These data support a role for ERRγ/AP1 signaling in the development of TAM resistance and suggest that expression of ERRγ may be a marker of poor TAM response. [Cancer Res 2008;68(21):8908–17]

Background: Multiple Endocrine Neoplasia (MEN) 2B is a rare hereditary disorder characterized by medullary thyroid cancer (MTC) in early childhood, pheochromocytoma, and mucosal neuromas. Patients with advanced MTC are treated with rearranged during transfection (RET) targeting tyrosine kinase inhibitors (TKIs) such as vandetanib. Despite initial responses, many patients progress on TKI therapy and mechanisms of resistance are yet to be elucidated. We analyzed tumor samples from seven children with MEN2B and MTC enrolled in a natural history study (NCT01660984). Methods: DNA samples from tumor and adjacent normal tissue from paraffin embedded blocks or unstained slides were analyzed by a custom capture next-generation sequencing panel. Bioinformatics analyses identified point mutations, insertions, deletions and copy number variations. Results: Seven patients (median age at study enrollment 14 years, range 11-17 years) with the RET p.Met918Thr germline mutation were included in analysis. Tumor samples were available for three patients pre-TKI (four samples), two patients at progression on TKI (three samples), and two patients both, pre-TKI and at progression (five samples). Pre-TKI samples exhibited few tumor specific mutations or copy number variations and 4/5 patients had loss of chromosome 1p. Progression for one patient was associated with acquisition of a previously unidentified p.Leu790Phe mutation within the kinase domain of the RET gene. Loss of heterozygosity and increase in copy number variations were noted in 4/5 samples at tumor progression. Two patients had copy number loss of chromosome 14 and three had copy number gain of chromosome 1q. Recurrent, somatic, non-synonymous mutations were not identified in the sample set. Conclusion: In children with MEN2B and MTC, we identified increase in copy number variations and a somatic mutation within the RET gene as potential mechanisms of drug resistance. Our data imply that a common genetic mechanism for progression on TKI may not exist within this small sample set and highlight the need for serial collection of tumor tissue. Further, whole genome aneuploidy may provide rationale for the evaluation of cytotoxic chemotherapy in patients who experience progressive disease on TKI therapy. Analysis of additional samples and whole exome DNA and RNA sequencing are ongoing. Citation Format: Ira L. Kraft, Srivandana Akshintala, Keith J. Killian, Robert B. Hufnagel, John W. Glod, Claudia Derse-Anthony, Yuelin Zhu, Holly S. Stevenson, Diana Bradford, Maria J. Merino, Frank M. Balis, Elizabeth Fox, Brigitte C. Widemann, Jack F. Shern, Paul S. Meltzer. Genomic mechanisms of disease progression in pediatric medullary thyroid cancer (MTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2017-4882

Circulating tumor cells (CTCs) are rare cancer cells in the bloodstream thought to have a key role in cancer metastasis. There are enormous interests in their analysis for castration-resistant prostate cancer (CRPC) due to their tendency to metastasize to bone. In this study, we applied a surface marker-independent microfluidic device for CTCs capture and retrieval that is based on cell size and deformability. Our device was able to capture 90% and recover 30% of tumor cells in spike experiments. We also developed and validated assays capable of detecting the transcript levels of selected molecular markers in single cells prepared with C1TM Single-Cell AutoPrep System. Blood samples drawn from 34 CRPC patients and 10 primary prostate cancer patients (PPC) were preceded for CTC isolation and molecular characterization. We found that 73% CRPC patients and 10% PPC patients were positive for EpCAM, and only about half of the EpCAM-positive CRPC cases were PSA or AR positive. It is interesting that while there is a strong association between AR and PSA expression in CTC samples, there is no correlation between AR or PSA transcripts in CTCs and serum PSA protein levels in CRPC patients. The positive detection of AR or PSA, but not EpCAM is statistically associated with poor prognosis of the CRPC patients. Very intriguingly, we identified a novel androgen receptor (AR) transcript variant -ARv from four CRPC patients’ CTCs. This ARv lacks a 554bp sequence region in the ligand binding domain (LBD), resulting in a loss of the entire LBD. Clinical data analysis revealed that the ARv is significantly associated with worse survival of CRPC patients (p Citation Format: Zhigang Kang, Avani Shah, Yunkai Yu, Yuelin Zhu, Ali Asgar Bhagat, Kyra Zhao, Andrew Wu, James Gao, Ravi Madan, James Gulley, William Dahut, Paul Meltzer, Liang Cao. A novel transcript variant of androgen receptor identified in circulating tumor cells from castration-resistant prostate cancer patients as a potentially prognostic biomarker. [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 366. doi:10.1158/1538-7445.AM2015-366

Circulating tumor cells (CTCs) are rare cancer cells circulating in the bloodstream that are thought to have a key role in cancer metastasis. They have been associated with worse prognosis in several major cancer types. There is an intense interest in the molecular analysis of CTC to gain information that would assist treatment decisions and potentially improve the disease outcome. There are enormous interests in the analysis of CTCs for castration-resistant prostate cancer (CRPC) due to the nature of the tumor that tends to metastasize to bone, making them generally inaccessible to obtain biopsies. In this study, we applied a surface marker independent, label-free cell trap microfluidics device for CTCs isolation and retrieval based on cell size and deformability. A total of 47 blood samples drawn from 36 patients with CRPC were preceded for CTC isolation followed by molecular characterization using epithelial tumor markers and prostate cancer specific markers. We identified a novel androgen receptor (AR) splice variant from four patients' CTCs, and confirmed by DNA sequencing that this variant lacks a 554bp region in the ligand binding domain of the receptor in comparison with wild type human AR. Patients with this splice variant were found to be associated with poor survival (p Note: This abstract was not presented at the meeting. Citation Format: Zhigang Kang, Yunkai Yu, Liang Cao, Paul S. Meltzer, Yuelin Zhu, Avan Shah, James Gulley, William Duht, Ravi Madan, Ali Bhagat, Kyra Zhao. A novel AR splice variant identified in circulating tumor cells from castration-resistant prostate cancer patient blood as a potentially prognostic biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4815. doi:10.1158/1538-7445.AM2014-4815

Although driver mutations in signal transduction kinases such as KIT are found in the majority of gastrointestinal stromal tumors (GIST), a subset of GISTs lack these mutations and instead exhibit loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle. To examine the effects of this metabolic defect on the epigenome, we used Illumina GoldenGate and 450K Infinium microarray technology to profile DNA methylation in GIST samples and uncovered markedly divergent global DNA methylation between SDH-null GIST (N=24) versus KIT or related tyrosine kinase pathway mutated GIST (N=39). When compared to reference normal tissues including intestinal smooth muscle (N=10) and neuronal tissue (N=13), SDH-deficient GIST was found to have an order of magnitude greater global hypermethylation than the kinase-pathway mutant group (84.9K vs. 8.4K targets, respectively). In a histologically distinct SDH-deficient tumor system, methylation divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (N=29) using an adrenal tissue (N=15) reference baseline. These data expose an essential role for succinate metabolism in the maintenance of DNA methylation programming and tumor suppression. Because defects in other Krebs cycle enzymes are also oncogenic, we further sought to determine whether this phenomenon was confined to SDH-deficient tumors. Analysis of SDH-mutant GIST and isocitrate dehydrogenase (IDH)-mutant gliomas revealed comparable quantities of global hypo- and hypermethylated targets. We propose that this phenomenon may result from a failure of maintenance demethylation, secondary to inactivation of the TET2 5-methylcytosine dioxgenase system by the inhibitory metabolites succinate (in SDH deficient tumors) or 2-hydroxyglutarate (in IDH mutant tumors). While the biological ramifications of this distortion of the epigenome remain to be elucidated, this study clearly implicates the Krebs cycle as mitochondrial custodian of the methylome in diverse cancers. Citation Format: Paul S. Meltzer, J. Keith Killian, Su Young Kim, Markku Miettinen, Carly Smith, Maria Tsokos, Martha Quezado, William I. Smith, Mona S. Jahromi, Robert L. Walker, Jerzy Lasota, Brandy Klotzle, Zengfeng Wang, Laura Jones, Yuelin Zhu, Yonghong Wang, Joshua J. Waterfall, Marina Bibikova, Maureen J. O'Sullivan, Constantine A. Stratakis, Joshua D. Schiffman, Jian-Bing Fan, Lee Helman. Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2013-2963

miRNAs are ∼22nt long non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level and play key roles in fundamental cellular processes. Deregulation of miRNAs has been observed in several diseases including cancer. Sarcomas are a heterogeneous group of rare mesenchymal malignancies with high rates of metastasis and local recurrence. A large variety of histotypes with very few reliable markers makes sarcoma diagnosis challenging. Hence, a refined classification scheme with novel diagnostic, prognostic and predictive markers would be clinically valuable. Additionally, tumor specific alterations in the miRNA expression profile may lead to the identification of miRNA targets important in sarcoma tumor biology. The diagnostic value of miRNA expression profiling was assessed in a mixed series of 161 sarcomas representing 12 histopathological subtypes, 5 normal smooth muscle, 4 normal skeletal muscle and 4 normal adipose tissues, using Agilent miRNA oligoarrays containing 470 human and 64 viral miRNAs. High-throughput miRNA sequencing (Illumina) was performed in a subset (n=10) of these tumors to validate results from the array-based expression profiling. An unsupervised cluster analysis performed on the 174 tumor and normal samples revealed groups based predominantly on diagnosis and tissue-type. All hemangiopericytomas and dermatofibrosarcomas, most rhabdomyosarcomas, Ewing's sarcomas, myxoid/round-cell liposarcomas, synovial sarcomas, leiomyosarcomas, osteosarcomas, normal smooth muscle, normal skeletal muscle and normal adipose tissue formed distinct clusters. About half of the pleomorphic/dedifferentiated/well-differentiated liposarcomas and fibrosarcomas and a quarter of the malignant peripheral nerve sheath tumors grouped together whereas most of the malignant fibrous histiocytomas clustered poorly. Discriminatory miRNAs were identified for the various subtypes (e.g., miR-146, miR-99 and miR-222 were highly expressed in the hemangiopericytomas, miR-375, miR-206 and miR-1 in the rhabdomyosarcomas, miR-181, miR-101 and miR-10 in the Ewing's sarcomas, miR-140-3p, miR-199 and miR-152 in osteosarcomas and miR-143, miR-145 and miR-28 in leiomyosarcomas). miRNAs that distinguished normal skeletal muscle from rhabdomysarcomas (e.g. miR-130, miR-133), normal smooth muscle from leiomyosarcomas (e.g. miR-142, miR-376) and normal adipose tissue from liposarcomas (e.g. miR-155, miR-365) were also identified. The results from sequencing were highly correlated with the results from the microarray analysis. Distinct miRNA expression profiles that correlate with lineage and tumor biology were identified not only in sarcomas with simple type-specific genetic alterations but also in the pleomorphic subtypes suggesting an important role for miRNAs in their tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3024.