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Purpose of the study: This study was aimed at identifying epigenome signature associated with risk of pediatric leukemia and uncovering molecular precursors of leukemia at birth in the blood of children before they develop the disease. Pediatric cancer is the leading cause of disease-related mortality in children and adolescents, with increasing incidence worldwide and lifelong sequelae in survivors. The most common form is leukemia, the causes of which are largely unknown. Growing evidence points to an origin in utero, when global redistribution of the epigenome modifications occurs driving tissue differentiation. Here, we sought to identify genome-wide differentially methylated genes at birth in newborns who later developed pediatric precursor B-cell ALL (pre-B ALL), compared with those who did not. Experimental procedures: Epigenome-wide DNA methylation was profiled in neonatal blood, with follow-up to pediatric pre-B ALL, using double-blinded analyses between prospective cohorts extending from birth to diagnosis and retrospective studies backtracking from clinical disease to birth. Validation was done using an independent technology and population (totaling 317 cases and 483 control) and complemented with pan-tissue methylation-stability (n=5,023 tissues; 30 types) and methylation-expression (n=2,294 tissues; 26 types) analyses. At diagnosis, methylation analysis was performed in leukemia tissues from pre-B ALL patients (n=644) with at least ten-year follow-up. Results: We found a limited number of loci (among which an imprinted tumor suppressor gene) as being significantly hypermethylated at birth in nested cases relative to controls in all tested populations, including European and Hispanic ancestries. Some DMRs were found to be stable over follow-up years after birth and across surrogate blood and target bone marrow tissues. Differential methylation was found to be associated with a change in gene expression and with worse pre-B ALL patient survival, supporting a functional and translational role for differential methylation. Conclusions: Our results provide proof-of-concept to detect at birth epigenetic alterations predisposing to childhood leukemia, reproducible in three continents and two ethnicities. DNA methylation alterations evident before diagnosis could be precursors of pediatric pre-B ALL development and actionable targets for risk assessment and prognosis. Citation Format: Akram Ghantous, Semira Gonseth Nusslé, Farah Nassar, Natalia Spitz, Alexei Novoloaca, Olga Krali, Ritu Roy, Shaobo Li, Maxime Caron, Lilys Lam, Peter Daniel Fransquet, John Casement, John Strathdee, Mark S. Pearce, Helen M. Hansen, Adam J. De Smith, Daniel Sinnett, Siri Eldevik Håberg, Jill McKay, Jessica Nordlund, Per Magnus, Terence Dwyer, Richard Saffery, Joseph Leo Wiemels, Monica Cheng Munthe-Kaas, Zdenko Herceg. Epigenome-wide DNA methylation alterations precede diagnosis since birth and affect prognosis of pediatric B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB362.

Glucocorticoids (GCs) are a central component of therapy for patients with T cell acute lymphoblastic leukemia (T-ALL), and although resistance to GCs is a strong negative prognostic indicator in T-ALL, the mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled in the frontline Children's Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cells were cultured in the presence of IL-7, a cytokine that is critical for normal T cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL-7 receptor (IL-7R) expression. In the presence of IL-7, this augmented downstream signal transduction, resulting in increased STAT5 transcriptional output and upregulation of the prosurvival protein BCL-2. Taken together, we showed that IL-7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL-7R/JAK/STAT5/BCL-2 axis.

Detection of disseminated tumor cells (DTCs) in bone marrow is an established negative prognostic factor. We isolated small pools of (~20) EPCAM-positive DTCs from early breast cancer patients for genomic profiling. Genome-wide copy number profiles of DTC pools (n = 45) appeared less aberrant than the corresponding primary tumors (PT, n = 16). PIK3CA mutations were detected in 26% of DTC pools (n = 53), none of them were shared with matched PTs. Expression profiling of DTC pools (n = 30) confirmed the upregulation of EPCAM expression and certain oncogenes (e.g., MYC and CCNE1), as well as the absence of hematopoietic features. Two expression subtypes were observed: (1) luminal with dual epithelial–mesenchymal properties (high ESR1 and VIM/CAV1 expression), and (2) basal-like with proliferative/stem cell-like phenotype (low ESR1 and high MKI67/ALDH1A1 expression). We observed high discordance between ESR1 (40%) and ERRB2 (43%) expression in DTC pools vs. the clinical ER and HER2 status of the corresponding primary tumors, suggesting plasticity of biomarker status during dissemination to the bone marrow. Comparison of expression profiles of DTC pools with available data from circulating tumor cells (CTCs) of metastatic breast cancer patients revealed gene expression signatures in DTCs that were unique from those of CTCs. For example, ALDH1A1, CAV1, and VIM were upregulated in DTC pools relative to CTCs. Taken together, analysis of pooled DTCs revealed molecular heterogeneity, possible genetic divergence from corresponding primary tumor, and two distinct subpopulations. Validation in larger cohorts is needed to confirm the presence of these molecular subtypes and to evaluate their biological and clinical significance., Genetics: two subtypes of disseminated tumor cells in bone marrow Tumor cells found in the bone marrow of women with early-stage breast cancer constitute molecularly distinct populations that could impact patient outcomes and treatment responses. Mark Jesus Magbanua and coworkers from the University of California, San Francisco, USA, performed genome-wide copy number variation analysis, expression profiling on 64 cancer-related genes and screened for PIK3CA mutations in pools of tumor cells isolated from bone marrow aspirates of patients undergoing surgery. These disseminated tumor cells (DTCs) contained fewer genomic abnormalities on average than their corresponding primary tumors, although the DTCs often acquired oncogenic mutations in PIK3CA. Expression profiling revealed two distinct subtypes of DTCs, both of which were different from circulating tumor cells found in the bloodstream. Further studies are needed to evaluate the biological and clinical significance of this molecular and genomic heterogeneity in DTCs.

Purpose: We profiled circulating tumor cells (CTCs) to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).Methods: CTCs were isolated from 105 patients with MBC using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACS), 28 of whom had serial CTC analysis (74 samples, 2–5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (n = 151) and genome-wide copy-number analysis by array comparative genomic hybridization (aCGH; n = 49).Results: Combined transcriptional and genomic profiling showed that CTCs were 26% ESR1−ERBB2−, 48% ESR1+ERBB2−, and 27% ERBB2+. Serial testing showed that ERBB2 status was more stable over time compared with ESR1 and proliferation (MKI67) status. While cell-to-cell heterogeneity was observed at the single-cell level, with increasingly stable expression in larger pools, patient-specific CTC expression “fingerprints” were also observed. CTC copy-number profiles clustered into three groups based on the extent of genomic aberrations and the presence of large chromosomal imbalances. Comparative analysis showed discordance in ESR1/ER (27%) and ERBB2/HER2 (23%) status between CTCs and matched primary tumors. CTCs in 65% of the patients were considered to have low proliferation potential. Patients who harbored CTCs with high proliferation (MKI67) status had significantly reduced progression-free survival (P = 0.0011) and overall survival (P = 0.0095) compared with patients with low proliferative CTCs.Conclusions: We demonstrate an approach for complete isolation of EPCAM-positive CTCs and downstream comprehensive transcriptional/genomic characterization to examine the biology and assess breast cancer biomarkers in these cells over time. Clin Cancer Res; 24(6); 1486–99. ©2018 AACR.

Author(s): Afshar, Armin R; Damato, Bertil E; Stewart, Jay M; Zablotska, Lydia B; Roy, Ritu; Olshen, Adam B; Joseph, Nancy M; Bastian, Boris C | Abstract: PurposeTo clinically use the UCSF500, a pancancer, next-generation sequencing assay in uveal melanoma (UM) and to correlate results with gene expression profiling (GEP) and predictive factors for metastasis.MethodsCohort study. Tumor samples of adult UM patients were analyzed with the UCSF500 and GEP. Main outcomes were copy number changes in chromosomes 1, 3, 6, and 8 and mutations in GNAQ, GNA11, SF3B1, EIF1AX, BAP1, SRSF2, U2AF1, and PLCB4. Chromosome 3 loss (a metastasis predictor) was tested for correlation with GEP class, tumor characteristics (largest basal diameter, thickness, ciliary body involvement, and extraocular extension), and histology (presence of epithelioid cells, closed loops, and mitotic count).ResultsThe 62 patients had a mean age of 59 years (range, 24-89 years). Chromosome 3 loss was detected in 30 patients and was associated with larger basal tumor diameter (Wilcoxon rank sum test, P = 0.015), greater thickness (Wilcoxon rank sum test, P = 0.016) and tumor, node, metastasis stage (Fisher test, P = 0.006), epithelioid cytology (Fisher test, P l 0.001), BAP1 mutation (Fisher test, P l 0.001), and chromosome 8q gain (Fisher test, P l 0.001). Class 2 tumors were much more likely to have chromosome 3 loss than class 1 (odds ratio, 121; P l 0.001). Eleven patients developed metastatic UM, of which five died during the study. All metastatic cases had chromosome 3 loss, 8 gain, BAP1 mutation, and class 2 GEP. Five class 1 tumors had chromosome 3 loss.ConclusionsUCSF500 detects chromosomal copy number changes and missense mutations that correlate strongly with metastasis predictors, including GEP.Translational relevanceNext-generation sequencing of UM should enhance survival prognostication.

10002 Background: The heterogeneity of T-ALL has hindered biomarker identification and limited biology-based risk stratification. Historically, minimal residual disease (MRD) has been the strongest predictor of poor outcomes. However, stratification by MRD does not allow for risk-adapted therapy early in treatment, which may induce deeper remissions and decrease risk of relapse. We hypothesized that gene expression profiling at diagnosis may have prognostic value in identifying high risk patients. Methods: We analyzed RNA-seq data from 189 diagnostic samples from the Children’s Oncology Group (COG) AALL0434 trial. Using leave-one-out cross-validation, we identified a set of genes that optimally differentiated MRD+ and MRD- samples. We then derived a risk score (RS) that indicates a probability of being MRD+ for a given gene expression pattern. Finally, we validated this model in an independent cohort of COG AALL1231 samples. Results: The AALL0434 early T-cell precursor (ETP) samples (n = 19), which have high rates of MRD+, had the highest RS, with an average of 81.3 (SD 18.7), versus 24.9 (SD 22.7) for non-ETPs (n = 146). Intriguingly, non-ETPs with RS > 50 had a gene expression pattern that mirrored ETPs and was distinct from the remaining non-ETPs. In this RS > 50 non-ETP cohort, 80% were MRD+, versus 20% of the < 50 cohort (p < 0.0001). When applied to 31 diagnostic non-ETP samples from COG AALL1231, 57% of the RS > 50 cohort were MRD+, versus 17% of the RS < 50 cohort (p = 0.05). Importantly, AALL0434 used prednisone during induction, while AALL1231 used dexamethasone, indicating that the predictive value is independent of the induction steroid. Finally, we converted our model to the customizable Nanostring nCounter platform by analyzing 96 AALL0434 samples on the Nanostring assay. The Nanostring data closely recapitulated the RNA-seq data, with a tight correlation between the resulting RS (concordance correlation coefficient = 0.91). Conclusions: We have developed a gene expression classifier that differentiates a subset of non-ETP T-ALLs with an ETP-like gene expression pattern and a high risk of MRD+, and have adapted the classifier to a clinically tractable targeted platform. Identification of this high-risk subset at diagnosis has the potential to facilitate risk-adapted trials to evaluate the utility of novel or more intensive therapies aimed at improving clinical outcomes.

Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR+ mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types.

PURPOSE: To evaluate the time course and association with survival of anatomic lesion volumes and diffusion imaging parameters for patients with newly diagnosed glioblastoma who were treated with radiation and concurrently with either temozolomide and enzastaurin (TMZ+enza cohort) or temozolomide, erlotonib, and bevaciumab (TMZ+erl+bev cohort). MATERIALS AND METHODS: Regions of interest corresponding to the contrast-enhancing and hyperintense lesions on T2-weighted images were generated. Diffusion-weighted images were processed to provide maps of apparent diffusion coefficient, fractional anisotropy, and longitudinal and radial eigenvalues. Histograms of diffusion values were generated and summary statistics calculated. Cox proportional hazards models were employed to assess the association of representative imaging parameters with survival with adjustments for age, Karnofsky performance status, and extent of resection. RESULTS: Although progression-free survival was significantly longer for the TMZ+erl+bev cohort (12.8 vs 7.3 months), there was no significant difference in overall survival between the two populations (17.0 vs 17.8 months). The median contrast-enhancing lesion volumes decreased from 6.3 to 1.9 cm3 from baseline to the postradiotherapy scan for patients in the TMZ+enza cohort and from 2.8 to 0.9cm3 for the TMZ+erl+bev cohort. Changes in the T2 lesion volumes were only significant for the latter cohort (26.5 to 11.9 cm3). The median apparent diffusion coefficient and related diffusion parameters were significantly increased for the TMZ+enza cohort (1054 to 1225 μm2/s). More of the anatomic parameters were associated with survival for the TMZ+enza cohort, whereas more diffusion parameters were associated with survival for the TMZ+erl+bev cohort. CONCLUSION: The early changes in anatomic and diffusion imaging parameters and their association with survival reflected differences in the mechanisms of action of the treatments that were being given. This suggests that integrating diffusion metrics and anatomic lesion volumes into the Response Assessment in Neuro-Oncology criteria would assist in interpreting treatment-induced changes and predicting outcome in patients with newly diagnosed glioblastoma who are receiving such combination treatments.

BACKGROUND Carotenoids are a class of nutrients with antioxidant properties that have been purported to protect against cancer. However, the reported associations between carotenoids and prostate cancer have been heterogeneous and lacking data on interactions with nucleotide sequence variations and genomic biomarkers. OBJECTIVE To examine the associations between carotenoid levels and the risk of high-grade prostate cancer, also considering antioxidant-related genes and tumor instability. METHODS We measured plasma levels of carotenoids and genotyped 20 single nucleotide polymorphisms (SNP) in SOD1, SOD2, SOD3, XRCC1, and OGG1 among 559 men with non-metastatic prostate cancer undergoing radical prostatectomy. We performed copy number analysis in a subset of these men (n = 67) to study tumor instability assessed as Fraction of the Genome Altered (FGA). We examined associations between carotenoids, genotypes, tumor instability and risk of high-grade prostate cancer (Gleason grade ≥ 4 + 3) using logistic and linear regression. RESULTS Circulating carotenoid levels were inversely associated with the risk of high-grade prostate cancer; odds ratios (OR) and 95% confidence intervals (CI) comparing highest versus lowest quartiles were: 0.34 (95% CI: 0.18–0.66) for α-carotene, 0.31 (95% CI: 0.15–0.63) for β-carotene, 0.55 (0.28–1.08) for lycopene and 0.37 (0.18–0.75) for total carotenoids. SNPs rs25489 in XRCC1, rs699473 in SOD3 and rs1052133 in OGG1 modified these associations for α-carotene, β-carotene and lycopene, respectively (P ≤ 0.05). The proportion of men with a high degree of FGA increased with Gleason Score (P

BACKGROUND: Although the contrast-enhancing (CE) lesion on T 1 -weighted MR images is widely used as a surrogate for glioblastoma (GBM), there are also non-enhancing regions of infiltrative tumor within the T 2 -weighted lesion, which elude radiologic detection. Because non-enhancing GBM (Enh−) challenges clinical patient management as latent disease, this study sought to characterize ex vivo metabolic profiles from Enh− and CE GBM (Enh+) samples, alongside histological and in vivo MR parameters, to assist in defining criteria for estimating total tumor burden. Methods: Fifty-six patients with newly diagnosed GBM received a multi-parametric pre-surgical MR examination. Targets for obtaining image-guided tissue samples were defined based on in vivo parameters that were suspicious for tumor. The actual location from where tissue samples were obtained was recorded, and half of each sample was analyzed for histopathology while the other half was scanned using HR-MAS spectroscopy. Results: The Enh+ and Enh− tumor samples demonstrated comparable mitotic activity, but also significant heterogeneity in microvascular morphology. Ex vivo spectroscopic parameters indicated similar levels of total choline and N -acetylaspartate between these contrast-based radiographic subtypes of GBM, and characteristic differences in the levels of myo-inositol, creatine/phosphocreatine, and phosphoethanolamine. Analysis of in vivo parameters at the sample locations were consistent with histological and ex vivo metabolic data. CONCLUSIONS: The similarity between ex vivo levels of choline and NAA, and between in vivo levels of choline, NAA and nADC in Enh+ and Enh− tumor, indicate that these parameters can be used in defining non-invasive metrics of total tumor burden for patients with GBM.

Background Differentially methylated regions (DMRs) within DNA isolated from whole blood can be used to estimate the proportions of circulating leukocyte subtypes. We use the term “immunomethylomics” to describe the application of these immune lineage DMRs to studying leukocyte profiles. Here, we applied this approach to peripheral blood DNA from 72 glioma patients with molecularly defined brain tumors, representing common patient groups with defined characteristic survival times and risk factors. We first estimated the proportions of leukocyte subtypes in samples using deconvolution algorithms with reference DMR libraries from isolated leukocyte populations and Illumina 450K DNA methylation data. Then, we calculated the neutrophil to lymphocyte ratio (NLR) using methylation-derived cell composition estimates (mdNLR). The NLR is considered an indicator of immunosuppressive cells in cancer patients. Results Elevated mdNLR scores were observed in glioma patients compared to mdNLR values of published controls. Significantly decreased survival times were associated with mdNLR ≥ 4.0 in Cox proportional hazards models adjusted for age, gender, tumor grade, and molecular subtype (HR 2.02, 95% CI, 1.11–3.69). We also identified five myeloid-related CpGs that were highly correlated with the mdNLR (adjusted R 2 ≥ 0.80). Each of the five myeloid CpG loci was associated with survival when adjusted for the above covariates and offer a simplified approach for utilizing fresh or archived peripheral blood samples for interrogating a very small number of methylation markers to estimate myeloid immune influences in glioma survival. Conclusions The mdNLR (based on DNA methylation) is a novel candidate methylation biomarker that represents immunosuppressive myeloid cells within the blood of glioma patients with potential application in clinical trials and future epidemiologic studies of glioma risk and survival. Electronic supplementary material The online version of this article (doi:10.1186/s13148-017-0316-8) contains supplementary material, which is available to authorized users.

While the cytogenetic and genetic characteristics of childhood acute lymphoblastic leukemias (ALL) are well studied, less clearly understood are the contributing epigenetic mechanisms that influence the leukemia phenotype. Our previous studies and others identified gene mutation (RAS) and DNA methylation (FHIT) to be associated with the most common cytogenetic subgroup of childhood ALL, high hyperdiploidy (having five more chromosomes). We screened DNA methylation profiles, using a genome-wide high-dimension platform of 166 childhood ALLs and 6 normal pre-B cell samples and observed a strong association of DNA methylation status at the PTPRG locus in human samples with levels of PTPRG gene expression as well as with RAS gene mutation status. In the 293 cell line, we found that PTPRG expression induces dephosphorylation of ERK, a downstream RAS target that may be critical for mutant RAS-induced cell growth. In addition, PTPRG expression is upregulated by RAS activation under DNA hypomethylating conditions. An element within the PTPRG promoter is bound by the RAS-responsive transcription factor RREB1, also under hypomethylating conditions. In conclusion, we provide evidence that DNA methylation of the PTPRG gene is a complementary event in oncogenesis induced by RAS mutations. Evidence for additional roles for PTPR family member genes is also suggested. This provides a potential therapeutic target for RAS-related leukemias as well as insight into childhood ALL etiology and pathophysiology.

Children with Down syndrome (DS), caused by constitutive trisomy of chromosome 21, have a 20-fold increased risk of acute lymphoblastic leukemia (DS-ALL) and 500-fold increased risk of acute megakaryoblastic leukemia (DS-AMKL). At least 10-15% of DS neonates are born with the pre-leukemic syndrome transient abnormal myelopoiesis (TAM). Trisomy 21 affects hematopoiesis and leukemia risk; however, the underlying mechanisms are not fully understood. Previous small-scale studies (sample N DNA was extracted from newborn dried bloodspots from 196 children born with DS and 442 non-DS controls from the California Biobank Program, and assayed using Illumina Infinium MethylationEPIC arrays containing >850,000 CpG probes. Data preprocessing was performed using the SeSAMe R package, and the conumee package confirmed trisomy 21 or euploidy in DS and non-DS subjects. Cell type deconvolution was performed, and blood cell proportions compared between DS and non-DS newborns using linear regression adjusting for covariates. ReFACTor principal components (PCs) were used to adjust for cell type heterogeneity and EPISTRUCTURE PCs to adjust for ancestry. Epigenome-wide association studies (EWAS) were performed overall, and stratified by ethnicity, using linear regression models adjusting for sex, plate, first six ReFACTor PCs, and first six ancestry-related PCs. Differentially methylated regions (DMRs) were evaluated using DMRcate and comb-p, with a consensus list of significant DMRs using the overlap. Deconvolution of blood cell proportions revealed highly significant (P Intriguingly, PC analysis and hierarchical clustering of DNA methylation data identified a subset of DS subjects (N=34/196, 17%) that clustered separately: these all had significantly higher nRBC proportions than other subjects and possibly represent DS neonates with TAM, although further investigation is needed for confirmation. Removing this cluster did not affect our main findings in our EWAS or DMR analyses. Constitutive trisomy 21 has profound effects on DNA methylation across the genome, in particular resulting in repression of known regulators of hematopoiesis including RUNX1 and FLI1. Our findings highlight potential mechanisms for the increased risk of both lymphoid and myeloid malignancies in children with DS. Disclosures No relevant conflicts of interest to declare.

10033 Background: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, which has largely precluded the use of genetic mutations for risk stratification. We hypothesized that despite this heterogeneity, diverse T-ALLs may have functional similarities that underlie patterns of chemotherapy sensitivity. Methods: We used flow cytometry to evaluate in vitro dexamethasone (DEX) sensitivity and baseline expression of signal transduction effectors and BCL2-family proteins in 68 fresh diagnostic T-ALL samples from patients enrolled on the Children’s Oncology Group (COG) trial AALL1231. We also performed RNA-sequencing (RNA-seq) on 40 AALL1231 samples and used hierarchical clustering and linear regression to analyze these and published T-ALL RNA-seq data from COG AALL0434. Comparisons between groups were made using t-tests and Fisher’s exact tests. Results: Of the proteins analyzed, only high BCL2 expression was significantly associated with increased in vitro DEX resistance (p = 0.002). Hierarchical clustering of the AALL1231 RNA-seq data identified two distinct clusters. Cluster 1 was associated with significantly higher BCL2 transcript expression (p = 0.0002) and in vitro DEX resistance (p = 0.04) relative to cluster 2. We defined a gene set consisting of the top 210 differentially expressed genes between these clusters and applied this gene set to the COG AALL0434 cohort. In this analysis, the early T-cell precursor (ETP) and near-ETP samples clustered together (p < 0.0001) in cluster 1 along with 39 of 146 non-ETP samples. Not only did these cluster 1 non-ETP samples have significantly higher BCL2 transcript expression relative to the non-ETP samples in cluster 2 (p < 0.0001), but 54% of these non-ETP samples were minimal residual disease (MRD) positive (≥0.01%) at the end of induction, as opposed to only 16% of the non-ETP samples in cluster 2 (p < 0.0001). Conclusions: Gene expression profiling identifies non-ETP T-ALLs that cluster with ETP/near-ETP T-ALLs and have significantly higher BCL2 expression and increased rates of post-induction MRD.

Although leptomeningeal carcinomatosis is a well-established clinical syndrome, virtually nothing is known about the tumor cells responsible for this particularly aggressive metastatic process. To isolate cerebrospinal fluid–derived tumor cells (CSFTC) from 15 patients with metastatic breast cancer diagnosed with leptomeningeal carcinomatosis, CSF samples were subjected to a two-step method involving immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS), a technique previously used for isolating circulating tumor cells (CTC) from blood. CSFTCs were subjected to genome-wide copy number analysis by array comparative genomic hybridization. Genomic profiling was successfully performed for 13 of 15 patients (87%). Copy number analysis in CSFTCs revealed genomic alterations commonly observed in primary breast cancer and CTCs, indicating their malignant origin. Interestingly, 12 (92%) harbored high-level gains on the 8q24 locus, which includes the MYC oncogene. Comparison of CSFTCs against corresponding archival primary tumors in six patients revealed clonal relationships with some divergence. Good concordance among serial samples attested to the reproducibility of the assay. Our approach for isolation and molecular analysis of CSFTCs yielded new insights into the molecular nature of these cells. Further genomic and functional analyses may help elucidate mechanisms by which tumor cells metastasize to the central nervous system. Cancer Res; 73(23); 7134–43. ©2013 AACR.

Interpretation of changes in the T1- and T2-weighted MR images from patients with newly diagnosed glioblastoma (GBM) treated with standard of care in conjunction with anti-angiogenic agents is complicated by pseudoprogression and pseudoresponse. The hypothesis being tested in this study was that 3D H-1 magnetic resonance spectroscopic imaging (MRSI) provides estimates of levels of choline, creatine, N-acetylaspartate (NAA), lactate and lipid that change in response to treatment and that metrics describing these characteristics are associated with survival. Thirty-one patients with newly diagnosed GBM and being treated with radiation therapy (RT), temozolomide, erlotinib and bevacizumab were recruited to receive serial MR scans that included 3-D lactate edited MRSI at baseline, mid-RT, post-RT and at specific follow-up time points. The data were processed to provide estimates of metrics representing changes in metabolite levels relative to normal appearing brain. Cox proportional hazards analysis was applied to examine the relationship of these parameters with progression free survival (PFS) and overall survival (OS). There were significant reductions in parameters that describe relative levels of choline to NAA and creatine, indicating that the treatment caused a decrease in tumor cellularity. Changes in the levels of lactate and lipid relative to the NAA from contralateral brain were consistent with vascular normalization. Metabolic parameters from the first serial follow-up scan were associated with PFS and OS, when accounting for age and extent of resection. Integrating metabolic parameters into the assessment of patients with newly diagnosed GBM receiving therapies that include anti-angiogenic agents may be helpful for tracking changes in tumor burden, resolving ambiguities in anatomic images caused by non-specific treatment effects and for predicting outcome.

Background: A major challenge in interpreting high-throughput multianalyte genomic data sets such as those produced by the ISPY clinical trials is data integration and interpretation within the context of biologically relevant pathways. To address this need, the data analysis tool PARADIGM (PAthway Recognition Algorithm using Data Integration on Genomic Models) was developed to infer the activities of genetic pathways by integrating any number of functional genomic data sets for a given patient sample into a pathway activity profile. Methods: We used PARADIGM to integrate gene expression (Agilent 44K) and DNA copy number data (AFFY 22K and 330K MIP) from 133 ISPY-1 patients into pathway component activity levels for approximately 1400 curated signal transduction, transcriptional and metabolic pathways superimposed onto a single non-redundant ‘SuperPathway'. These pathway activities then become the substrate for statistical analyses to identify pathways characterizing different breast cancer subtypes, as well as those associated with recurrence and response to neoadjuvant chemotherapy within breast cancer subgroups. To identify subtype-specific pathway activities, we used ANOVA for initial feature filtering followed by Tukey analysis with Benjamini Hochberg multiple testing correction. For other binary outcome comparisons we used Mann-Whitney (2-sample Wilcoxon) analysis. PARADIGM results were corroborated with pathway enrichment analysis and filtered for significance. Results: In agreement with breast cancer cell line and other prior studies, basal-like and triple negative cancers are dominated by upregulation of the FOXM1 and MYC/Max subnetworks and downregulation of the FOXA1/ER signal transduction pathway, the converse of the activity pattern seen in luminal breast cancers. These and other subtype associations pass stringent multiple testing corrected significance tests. Though an association study of recurrence over the entire patient cohort mostly yields pathways characteristic of basal-like tumors, alternative pathway associations emerge when subtypes are analyzed individually for outcome and significance tests are relaxed to include features that pass un-corrected Wilcoxon significance tests and also generate highly significant pathway enrichment scores. Subtype-specific drivers of recurrence and chemo-resistance supported by this level of evidence include ALK1/2 (TGFB-BMP) and p53 effector signaling for basals and Syndecan-1 and c-MYC for luminals. Chemo-sensitivity pathways, assessed by association with pCR and RCB1, appear to be subtype-specific as well, with HDAC class 1 signaling, LRP6-Wnt, and IRE1alpha chaperones dominating basal-like cancers and c-MYB activity dominating Her2+ cancers, whereas chemo-sensitivity of HR+Her2- cancers though rare appears to be driven by the DNA damage axis (BRCA/BARD1). Conclusion: These and other similar analyses suggest that patients with TN or basal-like disease might benefit from the addition of ALK1 pathway inhibitors to treatment, whereas high risk HR+ patients might benefit from Syndecan-1 inhibitors. C-MYC/MAX inhibitors might benefit all high risk patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-09.

Melanoma is comprised of biologically distinct subtypes. The defining clinical, histomorphologic, and molecular features are not fully established. This study sought to validate the association between genetic and histomorphologic features previously described and to determine their reproducibility and association with important clinical variables. Detailed clinical and histomorphologic features of 365 primary cutaneous melanomas were assessed by 11 pathologists and correlated with mutation status of BRAF and NRAS. There was substantial agreement in the quantitative assessment of histomorphologic features showing similar or better interobserver reproducibility than the established World Health Organization classification scheme. We confirmed that melanomas with BRAF mutations showed characteristic morphologic features (P < 0.0001) and metastasized more frequently to regional lymph nodes (P = 0.046). Importantly, melanomas without mutations were a heterogeneous group, with a subset having very similar clinical and morphological features as those with BRAF mutation raising the possibility that they are biologically related. Our study confirms an association between histomorphologic features, mutation status, and pattern of metastasis, providing criteria for a refined melanoma classification aimed at defining biologically homogeneous disease subgroups.

BACKGROUND: The presence of disseminated tumor cells (DTCs) in bone marrow of early breast cancer (EBC) patients is a strong predictor of poor prognosis. We assessed heterogeneity in copy number, PIK3CA mutation, and gene expression in DTCs to shed light on the molecular biology of these cells. METHODS: We isolated EPCAM-positive DTCs in 71 EBC patients using immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). Isolated DTCs, along with corresponding primary tumors (n=16), were subjected to genome-wide copy number profiling (n=47) by array comparative genomic hybridization, and mutation screening of the PIK3CA gene (n=53) by Sanger sequencing. The expression of 64 cancer-related genes in DTCs was analyzed by microfluidic-based multiplexed RT-QPCR (n=30). DTC expression profiles were compared with available gene expression data from circulating tumor cells (CTCs) of metastatic breast cancer patients. RESULTS: Copy number profiles of DTCs were less aberrant and distinct from their corresponding primary tumors. PIK3CA mutations detected in 26% of DTCs were mutually exclusive to those found in matched primary tumors. Expression profiles of DTCs were distinguishable from marrow leukocytes, and displayed up-regulation of oncogenes MYC and CCNE1. Unsupervised hierarchical clustering analysis revealed two subtypes of DTCs: (1) luminal with dual epithelial-mesenchymal properties (high ESR1 and VIM/CAV1 expression), and (2) basal-like with proliferative/stem cell-like phenotype (low ESR1 and high MKI67/ALDH1A1 expression). DTCs possessed gene expression signatures that were unique from those of CTCs. ALDH1A1, CAV1 and VIM were up-regulated in DTCs relative to CTCs. ESR1/ER and ERBB2/HER2 status in DTCs vs. corresponding primary tumors showed high discordance (40% and 43%, respectively), suggesting shift in biomarker status in micrometastatic cells in the bone marrow. CONCLUSIONS: We demonstrate the feasibility of isolation and comprehensive molecular characterization of DTCs from bone marrow of EBC patients. Comparative genomic analysis suggests that DTCs disseminate early and acquire genomic aberrations independently of the primary tumor. Expression profiling revealed two distinct subpopulations of DTCs. Validation in larger cohorts is needed to confirm the presence of these molecular subtypes and to evaluate their biological and clinical significance. Citation Format: Mark Jesus M. Magbanua, Hope Rugo, Louai Hauranieh, Ritu Roy, Janet Scott, Jen Chieh Lee, Feng Hsiao, Eduardo Sosa, Denise Wolf, Laura van't Veer, Laura Esserman, John Park. Genomic and expression profiling reveals molecular heterogeneity of disseminated tumor cells in early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5578.

Background: CTCs offer a relatively non-invasive source of metastatic tissue for molecular analysis. To elucidate the underlying biology of blood-borne metastasis, we profiled CTCs from MBC patients (pts). Methods: CTCs were isolated by IE/FACS (immunomagnetic enrichment/fluorescence-activated cell sorting). Expression of 64 cancer-related genes in CTCs was analyzed via microfluidic-based multiplex QPCR. Genome-wide copy number (CN) analysis by array comparative genomic hybridization (ACGH) was performed on CTCs isolated from the same tumor-enriched blood samples. The Illumina platform was utilized for next generation sequencing and data was analyzed using NantOmics analysis pipeline and Nexus 8.0 software. Mutations were confirmed by Sanger sequencing or by digital droplet PCR. Results: Expression profiles of CTCs from 105 MBC pts clustered away from those of blood, indicating high-purity isolation of CTCs by IE/FACS. In addition to EPCAM, tumor-related genes, e.g., CCND1, MUC1, and TTF3 were upregulated in CTCs. Approximately 70% of the CTC samples were considered ER-positive, of which 47% were ER+HER2+, and 22% ER+HER2-. Among the ER+HER2- samples, about two-thirds (68%) had low proliferative (MKI67) status. HER2-positive and triple-negative CTCs accounted for 27% and 30% of the samples, respectively. Furthermore, 30% of the samples were assigned to luminal A, 6% to luminal B, 13% to Her2-enriched, 33% to basal-like, and 12% to normal-like subtypes. Expression profiling of CTCs in 74 serial blood samples from 28 pts showed fluctuations in expression at the gene-level, while subtype calls were mostly consistent across time points. CTCs from 49 of the 105 pts analyzed by ACGH revealed numerous genomic aberrations such as 1q/8q gains and 8p/16q losses, consistent with breast cancer origin. CN profiles grouped into three major clusters: CTCs exhibiting low genomic instability, 8q gain, and 1q gain/11q loss. ERBB2 and CCND1 were upregulated in CTCs showing increased genomic alterations. Changes in ER (n=102) and HER2 (n=130) status between CTCs and matched primary tumors (PT) were observed in 27% and 23% of the pts, respectively, indicating that biomarker status may change during disease progression. Comparative analysis of CN data from low-pass whole genome sequencing (WGS) of CTCs vs. matched PTs (n=7 pairs) demonstrated clonal-relatedness as well as some genetic divergence. WGS (38x) and whole exome sequencing (140x) analysis of CTCs from an index pt diagnosed with invasive lobular carcinoma detected numerous genomic aberrations, including a copy loss and a frameshift mutation in E-cadherin (CDH1). Interestingly, analysis of CN and mutation data revealed that CTCs were more closely related to the lymph node metastases than to the PT. Single-cell sequencing of CTCs revealed uniformity in genome-wide CN alterations, while cell-to-cell heterogeneity was observed only when single-cell expression profiles were analyzed. Conclusions: Comprehensive molecular characterization provided novel insights into the biology of breast CTCs. Further CTC profiling may open avenues for discovery and development of novel biomarkers for personalized medicine and strategies to prevent metastasis. Citation Format: Magbanua MJ, Hauranieh L, Roy R, Wolf D, Benz S, Vaske C, Pendyala P, Sosa E, Scott J, Lee JS, Ordonez A, Ho B, Solanki T, VantVeer L, Rugo H, Park J. Comprehensive genomic characterization of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) sheds light on the biology of blood-borne metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-04.

Background The heterogeneous biology of glioblastoma (GBM) emphasizes the need for imaging methods to assess tumor burden and assist in evaluating individual patients. The purpose of this study was to investigate early changes in metrics from 3D 1H magnetic resonance spectroscopic imaging (MRSI) data, compare them with anatomic lesion volumes, and determine whether they were associated with survival for patients with newly diagnosed GBM receiving a multimodality treatment regimen. Methods Serial MRI and MRSI scans provided estimates of anatomic lesion volumes and levels of choline, creatine, N-acetylaspartate, lactate, and lipid. The association of metrics derived from these data with survival was assessed using Cox proportional hazards models with adjustments for age, Karnofsky performance score, and extent of resection. Temporal changes in parameters were evaluated using a Wilcoxon signed rank test. Results Anatomic lesion volumes at the post-radiotherapy (RT) scan, metabolic lesion volume at mid-RT and post-RT scans, as well as metrics describing levels of choline, lactate, and lipid were associated with overall survival. There was a significant reduction in the enhancing lesion volume, increase in T2 lesion volume from mid-RT to post-RT, and decrease in parameters describing metabolite levels during these early time points. Conclusion The MRSI data provided metrics that described the effects of treatment on the metabolic lesion burden and were associated with overall survival. This suggests that adding these parameters to standard assessments of changes in anatomic lesion volumes could contribute to making early decisions about the efficacy of such combination therapies.

4020Background: Extrapulmonary poorly differentiated neuroendocrine carcinomas (EP-NEC) occur in nearly any site. Traditionally treated like small cell lung carcinoma (SCLC), optimal therapy has not yet been defined. We examined genomic alterations in EP-NEC of different sites in comparison to SCLC and to each other. Methods: Genomic profiling was performed on 867 NEC, including 593 SCLC and 274 EP-NEC (pathologist-confirmed poorly differentiated morphology) arising in the gastrointestinal (GI) tract and pancreas: 123 pancreas, 92 colon, and 59 “other GI” (esophageal, stomach, small intestine). Hybridization-captured, adaptor ligation based libraries were used to a mean coverage depth of 600X for 192 cancer-related genes. All classes of genomic alterations were identified (copy number losses, amplifications, rearrangements, and short variant mutations). Results: There were 9 genes with alterations in > 15% of tumors in any group (Table 1). Only TP53 crossed the 15% threshold in every group; MEN1 and DAXX ...

Molecular characterization of circulating tumor cells (CTC) from blood is technically challenging because cells are rare and difficult to isolate. We developed a novel approach to isolate CTCs from blood via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE–FACS). Isolated CTCs were subjected to genome-wide copy number analysis via array comparative genomic hybridization (aCGH). In clinical studies, CTCs were isolated from 181 patients with metastatic breast cancer, 102 of which were successfully profiled, including matched archival primary tumor from five patients. CTCs revealed a wide range of copy number alterations including those previously reported in breast cancer. Comparison with two published aCGH datasets of primary breast tumors revealed similar frequencies of recurrent genomic copy number aberrations. In addition, serial testing of CTCs confirmed reproducibility and indicated genomic change over time. Comparison of CTCs with matched archival primary tumors confirmed shared lineage as well as some divergence. We showed that it is feasible to isolate CTCs away from hematopoietic cells with high purity through IE–FACS and profile them via aCGH analysis. Our approach may be used to explore genomic events involved in cancer progression and to monitor therapeutic efficacy of targeted therapies in clinical trials in a relatively noninvasive manner. Cancer Res; 73(1); 30–40. ©2012 AACR.

Array comparative genomic hybridization was used to identify copy number alterations in clear cell renal cell carcinoma (ccRCC) patient tumors to identify associations with patient/clinical characteristics. Of 763 ccRCC patients, 412 (54%) provided frozen biopsies. Clones were analyzed for significant copy number differences, adjusting for multiple comparisons and covariates in multivariate analyses. Frequent alterations included losses on: 3p (92.2%), 14q (46.8%), 8p (38.1%), 4q (35.4%), 9p (32.3%), 9q (31.8%), 6q (30.8%), 3q (29.4%), 10q (25.7%), 13q (24.5%), 1p (23.5%) and gains on 5q (60.2%), 7q (39.6%), 7p (30.6%), 5p (26.5%), 20q (25.5%), 12q (24.8%), 12p (22.8%). Stage and grade were associated with 1p, 9p, 9q, 13q and 14q loss and 12q gain. Males had more alterations compared with females, independent of stage and grade. Significant differences in the number/types of alterations were observed by family cancer history, age at diagnosis and smoking status. Von Hippel-Lindau (VHL) gene inactivation was associated with 3p loss (PE-05), and these cases had fewer alterations than wild-type cases. The fragile site flanking the FHIT locus (3p14.2) represented a unique breakpoint among VHL hypermethylated cases, compared with wild-type cases and those with sequence changes. This is the first study of its size to investigate copy number alterations among cases with extensive patient, clinical/risk factor information. Patients characterized by VHL wild-type gene status (vs sequence alterations) and male (vs female) cases had more copy number alterations regardless of diagnostic stage and grade, which could relate to poor prognosis. © 2012 Macmillan Publishers Limited. All rights reserved.

Progression of primary prostate cancer to castration-resistant prostate cancer (CRPC) is associated with numerous genetic and epigenetic alterations that are thought to promote survival at metastatic sites. In this study, we investigated gene copy number and CpG methylation status in CRPC to gain insight into specific pathophysiologic pathways that are active in this advanced form of prostate cancer. Our analysis defined and validated 495 genes exhibiting significant differences in CRPC in gene copy number, including gains in androgen receptor (AR) and losses of PTEN and retinoblastoma 1 (RB1). Significant copy number differences existed between tumors with or without AR gene amplification, including a common loss of AR repressors in AR-unamplified tumors. Simultaneous gene methylation and allelic deletion occurred frequently in RB1 and HSD17B2, the latter of which is involved in testosterone metabolism. Lastly, genomic DNA from most CRPC was hypermethylated compared with benign prostate tissue. Our findings establish a comprehensive methylation signature that couples epigenomic and structural analyses, thereby offering insights into the genomic alterations in CRPC that are associated with a circumvention of hormonal therapy. Genes identified in this integrated genomic study point to new drug targets in CRPC, an incurable disease state which remains the chief therapeutic challenge. Cancer Res; 72(3); 616–25. ©2011 AACR.

Purpose: Problems in management of oral cancers or precancers include identification of patients at risk for metastasis, tumor recurrence, and second primary tumors or risk for progression of precancers (dysplasia) to cancer. Thus, the objective of this study was to clarify the role of genomic aberrations in oral cancer progression and metastasis. Experimental Design: The spectrum of copy number alterations in oral dysplasia and squamous cell carcinomas (SCC) was determined by array comparative genomic hybridization. Associations with clinical characteristics were studied and results confirmed in an independent cohort. Results: The presence of one or more of the chromosomal aberrations +3q24-qter, -8pter-p23.1, +8q12-q24.2, and +20 distinguishes a major subgroup (70%–80% of lesions, termed 3q8pq20 subtype) from the remainder (20%–30% of lesions, non-3q8pq20). The 3q8pq20 subtype is associated with chromosomal instability and differential methylation in the most chromosomally unstable tumors. The two subtypes differ significantly in clinical outcome with risk for cervical (neck) lymph node metastasis almost exclusively associated with the 3q8pq20 subtype in two independent oral SCC cohorts. Conclusions: Two subtypes of oral lesions indicative of at least two pathways for oral cancer development were distinguished that differ in chromosomal instability and risk for metastasis, suggesting that +3q,–8p, +8q, and +20 constitute a biomarker with clinical utility for identifying patients at risk for metastasis. Moreover, although increased numbers of genomic alterations can be harbingers of progression to cancer, dysplastic lesions lacking copy number changes cannot be considered benign as they are potential precursors to non-3q8pq20 locally invasive, yet not metastatic oral SCC. Clin Cancer Res; 17(22); 7024–34. ©2011 AACR.

Background Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial. Methods Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients. Results Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001). Conclusions We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients. Trial Registration ClinicalTrials.gov NCT00402285

Purpose: The three main treatment options for primary prostate cancer are surgery, radiation, and active surveillance. Surgical and radiation intervention for prostate cancer can be associated with significant morbidity. Therefore, accurate stratification predictive of outcome for prostate cancer patients is essential for appropriate treatment decisions. Nomograms that use clinical and pathologic variables are often used for risk prediction. Favorable outcomes exist even among men classified by nomograms as being at high risk of recurrence. Experimental Design: Previously, we identified a set of DNA-based biomarkers termed Genomic Evaluators of Metastatic Prostate Cancer (GEMCaP) and have shown that they can predict risk of recurrence with 80% accuracy. Here, we examined the risk prediction ability of GEMCaP in a high-risk cohort and compared it to a Kattan nomogram. Results: We determined that the GEMCaP genotype alone is comparable with the nomogram, and that for a subset of cases with negative lymph nodes improves upon it. Conclusion: Thus, GEMCaP shows promise for predicting unfavorable outcomes for negative lymph node high-risk cases, where the nomogram falls short, and suggests that addition of GEMCaP to nomograms may be warranted. Clin Cancer Res; 16(1); 195–202

Desmoplastic melanomas (DMs) comprise 4% of the overall melanoma burden and have a 5-year survival rate of 85%. DMs are dermal tumors characterized by spindled melanocytes situated within abundant desomplastic stroma. These unusual histological features commonly lead to misdiagnosis. Currently, there are no known genetic drivers. A better understanding of the underlying biology of desmoplastic melanoma would provide biomarkers and therapeutic opportunities. Towards this goal, we performed low-coverage genome and high-coverage exome sequencing of 20 DMs in a discovery cohort, followed by targeted sequencing of 293 candidate genes on a validation cohort of 42 cases. Additionally, high-resolution aCGH was performed on samples from both cohorts. A high mutation burden (median 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers sequenced to date. Mutation patterns strongly indicate that UV-radiation is the dominant mutagen and implicate a superficially located cell of origin despite their predominantly intradermal presentation. Novel alterations included recurrent promoter mutations and amplification of NF-kappa B inhibitor epsilon, NFKBIE (IkBϵ) in 14.5% of samples. The promoter mutations typically affect both alleles and occur over a highly conserved DNA region. The mutations are predicted to disrupt a canonical Ets Like Factor 1 (ELF1) binding site. In total, these data imply aberrant NF-kappa B signaling as a pathogenic feature of desmoplastic melanoma. Commonly mutated oncogenes in melanomas, in particular BRAF V600E and NRAS Q61K/R, were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS, and PIK3CA. Rb and p53 pathway alterations occurred respectively in 71% and 66% of tumors, affecting RB1, FBXW7, CDK4, PPP6C, CCND1, CDKN2A, TP53, and MDM2. Finally, TERT promoter mutations or amplifications occurred in 90% of tumors. The consequences of the mutations on protein expression levels was confirmed by immunostaining for NF1, EGFR, Rb, CDK4, CCND1, p16, p53, and Mdm2. Collectively, many of these oncogenic mutations are potentially druggable. In conclusion, desmoplastic melanomas harbor distinct genetic alterations that explain their unique biology, and this study illuminates genetic biomarkers and nominates targets for therapeutic intervention. Citation Format: Alan H. Shain, Maria Garrido, Thomas Botton, Eric Talevich, Iweh Yeh, Zack Sanborn, Jongsuk Chung, Nicholas Wang, Hojabr Kakavand, Graham Mann, John Thompson, Thomas Wiesner, Ritu Roy, Adam Olshen, Alexander Gagnon, Joe Gray, Nam Huh, Joe Hur, Klaus Busam, Richard Scolyer, Raymond Cho, Rajmohan Murali, Boris Bastian. Exome sequencing of desmoplastic melanoma reveals recurrent NFKBIE promoter mutations and diverse MAPK/PI3K pathway activating mutations. [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 2968. doi:10.1158/1538-7445.AM2015-2968

Introduction Previously, residential dust content of polycyclic aromatic hydrocarbons (PAHs) was associated with an increased risk of acute lymphoblastic childhood leukemia (ALL), and PAHs exposure levels have been associated with differentially methylated regions (DMRs) in candidate genes studies. Thus, we hypothesized that in utero exposure to residential PAHs may be associated with genome-wide DMRs in genes that may influence ALL development. Methods PAHs were measured in dust samples collected using a high-volume surface sampler or household vacuum cleaners from homes of California Childhood Leukemia Study participants who have lived in the same home since diagnosis. The different PAHs were summed according to their toxic equivalency. DNA was extracted from archived neonatal blood spots of participants, then treated with bisulfite and assayed on Illumina Infinium 450K genome-wide DNA methylation arrays. Following removal of cross-reacting probes, SNP-related and polymorphic CpGs, we analyzed the association of residential PAH levels with methylation intensity of 319,265 CpGs in two independent case-only datasets (model #1: set 1 cases n = 84, set 2 cases n = 51). We used a false discovery rate with random resampling to reduce the number of false-positive findings and cross-validated the results between sets. This first step of the analysis gave 11 significant and sign concordant CpGs. Then we assessed interactions between case/control status and PAHs levels with methylation intensities at those 11 CpGs in set 1 (model #2: total cases and controls n = 306). Models were adjusted for cell mixture, sex, gestational age, race and the first 2 principal components of the models’ residuals. Results In model #1, methylation levels at 11 CpGs were significantly associated with residential PAH levels and sign concordant in both sets with a corresponding q-value < 10−08. In model #2, the interaction terms between PAH and case/control status were significant (p Conclusions For the first time, our genome-wide methylation investigation found that residential PAH levels were associated with 11 CpGs at birth among children who later developed ALL. Moreover, the future cases had significantly different DNA methylation responses to PAH exposures than the unaffected controls for 7 CpGs. This may suggest a case/control difference present at birth in the sensitivity to PAHs in particular cell-cycle and tumor-suppressor genes. These CpGs may be good candidates to further investigate with respect to leukemogenesis. Citation Format: Semira Gonseth, Todd P. Whitehead, Ritu Roy, E. Andres Houseman, Adam J. de Smith, Mi Zhou, Seung-Tae Lee, Margaret R. Wrensch, Stephen M. Rappaport, Catherine Metayer, Joseph L. Wiemels. In utero polycyclic aromatic hydrocarbons exposure and genome-wide DNA methylation modifications at birth in children who develop acute lymphoblastic leukemia. [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 831. doi:10.1158/1538-7445.AM2015-831

Background: Recent enumeration studies on CTCs have demonstrated the clinical value of these cells. The molecular biology of CTCs, however, remains poorly understood. Methods: We performed RNA and DNA profiling of CTCs from metastatic breast cancer (MBC) patients. 244 blood samples from 162 MBC patients were subjected to immunomagnetic enrichment and fluorescence activated cell sorting (IE/FACS) to isolate highly pure CTCs. Microfluidic-based multiplex QPCR analysis was utilized to determine the expression levels of 64 cancer-related genes. Parallel genome-wide copy number analysis by array comparative genomic hybridization was also performed on CTCs isolated from the same enriched blood samples. Results: Transcriptional analysis of CTCs was successfully performed in 151 samples from 105 patients. We observed the up-regulation of several genes including CCND1, EPCAM, KRT7 and MUC1 and the down-regulation of hematopoietic-related genes including PTPRC (CD45) and CD68 (adjusted p Conclusions: Parallel gene expression and copy number profiling provided novel insights on the biology of CTCs. Molecular characterization of CTCs may shed further light on the biology of metastasis and disease progression. Citation Format: Mark Jesus Mendoza Magbanua, Louai Hauranieh, Ritu Roy, Praveen Pendyala, Eduardo Sosa, Janet Scott, Hope Rugo, John Park. Comprehensive molecular profiling of circulating tumor cells from metastatic breast cancer patients. [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 1591. doi:10.1158/1538-7445.AM2015-1591

Introduction Folate supplementation during pregnancy was previously associated with a lower risk of acute lymphoblastic childhood leukemia (ALL). Folate is involved in the “one-carbon” metabolic cycle necessary for shuttling methyl groups for DNA methylation. Given the observation that aberrant DNA methylation patterns are a characteristic of leukemic cells during ALL oncogenesis, we hypothesized that (1) low folate exposure during fetal development might modify the neonate's DNA genome-wide methylation pattern, and that (2) such a modified pattern can lead to ALL development. Here we investigate the first part of this hypothesis. Methods We analyzed healthy control participants of the California Childhood Leukemia case-control study. Participants’ peri-conception folate exposure was estimated from self-reported maternal folate intake and supplements, which was derived from a food frequency questionnaire on the diet in the year prior to pregnancy, as a surrogate for nutrient “environment” during the peri-conception period. DNA obtained from archived neonatal blood spots was purified, treated with bisulfite and assayed on the Illumina Infinium 450K genome-wide DNA methylation array. After removing cross-reacting probes, SNP-related and polymorphic CpGs, we analyzed the association of folate with methylation intensity of 319,265 CpGs in two independent datasets (n set 1 = 167 and n set 2 = 176). Models were adjusted for cell mixture, sex, gestational age, and race. We used a false discovery rate with random resampling to reduce the number of false-positive findings and we cross-validated the results between sets. Results Maternal folate intake was broadly associated with less child's DNA methylation: with a q-value Conclusions This study was the first to investigate the association between maternal folate intake during the peri-conception period and DNA methylation at birth with a genome-wide approach and it found numerous associations throughout the genome. The relationships between folate-sensitive CpG sites and DNA methylation events critical to childhood leukemogenesis will be considered in our subsequent research. Citation Format: Semira Gonseth, Ritu Roy, E. Andres Houseman, Adam de Smith, Mi Zhou, Seung-Tae Lee, Sébastien Nusslé, Amanda W. Singer, Margaret R. Wrensch, Catherine Metayer, Joseph L. Wiemels. Maternal folate intake at periconception and genome-wide DNA methylation modifications at birth in children. [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 2763. doi:10.1158/1538-7445.AM2015-2763

11048 Background: Isolation, enumeration, and genomic profiling of CRPC CTCs offers the potential to discover genetic changes that occur in advanced disease. The Vitatex VitaCap platform captures CTCs based on their ability to invade a collagenous matrix (CAM), allows for capture of invasive CTCs (iCTCs) independent of EpCAM status, and yields viable cells suitable for comprehensive genomic study. Here we sought to compare CTC yields between the CAM and CellSearch platforms, to determine the utility of prostate-specific membrane antigen (PSMA) as an iCTC biomarker, to identify iCTC clusters and iCTCs expressing stem-like markers, and to explore the feasibility of iCTC epigenomic analysis. Methods: CTCs were isolated and enumerated simultaneously using the CellSearch and CAM platforms in 23 men with mCRPC. CAM-isolated iCTCs were defined as EpCAM+PSMA+ and were enumerated immunocytochemically (ICC) and by flow cytometry. iCTC clusters were enumerated by ICC. The Illumina Infinium HumanMethylation27 BeadChip was used to determine whole genome methylation status for CAM isolated cells. Results: 35 samples were collected for CAM analysis. A median of 27 (range 0-800) and 23 (range 2-390) iCTCs/ml were detected by ICC and flow respectively. In a subset of 20 samples, a median of 7 CTCs/ml (range 0-85) were detected by the CellSearch platform. CTCs were detectable by either CAM or CellSearch in >95% of samples. iCTC clusters were observed in 23% of samples with a median 7 clusters/ml (range 1-200). iCTCs expressing stem-like markers CD44 and Seprase were detected in 70% and 97% of samples by ICC and flow respectively, with a median of 9/ml (range 1-264) by flow. The iCTC methylation profile highly resembled mCRPC. Conclusions: The CAM and CellSearch platforms yield comparable CTC counts. iCTC clusters and iCTCs expressing stem-like markers are detectable using the CAM platform, and the iCTC methylome closely resembles that of mCRPC. Correlation with clinical data may yield further insight into the functional significance of these findings.

The biologic behavior of oral dysplasia (precancers) and squamous cell carcinoma (SCC) is unpredictable, the main problems in management being identification of patients at risk for metastasis or risk of progression of dysplasias to cancer. Neck metastasis, the most significant clinical factor causing death in oral SCC, reduces survival by 50%. We utilized array comparative genomic hybridization (CGH) to clarify the role of genomic aberrations in oral cancer progression and metastasis. Two oral SCC cohorts: cohort#1, n=89 (Snijders et al, 2005)) and cohort#2, n=63 (with associated pathologic nodal status and 5 year clinical follow up) were profiled using BAC CGH arrays. Two dysplasia cohorts, one with no association to cancer (cohort D1, n=29) and one associated with previous or subsequent cancer (cohort D2, n=10) were also profiled for recurrent copy number aberrations using array CGH. Associations with clinical characteristics were studied and results confirmed in an independent SCC cohort (cohort#3, n=14, Smeets et al., 2009). All samples used in this study were restricted to oral cavity sites only and were archival formalin fixed paraffin embedded tissue. Copy number alterations distinguished two oral dysplasia and cancer subtypes. The presence of one or more of the chromosomal aberrations +3q24-qter, -8pter-p23.1, +8q12-q24.2, and +20 distinguished a major subgroup (70%-80% of lesions, termed 3q8pq20 subtype) from the remainder (20%-30% of lesions, non-3q8pq20). Most notably, 3q8pq20 and non-3q8pq20 subtypes differed significantly in clinical outcome with risk for cervical (neck) lymph node metastasis almost exclusively associated with the 3q8pq20 subtype in two independent oral SCC cohorts (cohort#2 and cohort#3). As metastasis to the cervical lymph nodes is a major determinant of survival in oral SCC patients, our findings indicate that chromosomal aberrations +3p, -8p, +8q and +20 provide a potential biomarker to identify patients at no or low risk of metastasis. The negative predictive value (NPV) i.e. the ability to predict N0 cases at biopsy was 93% for cohort#2 and 100% in cohort#3. Currently, almost all patients diagnosed with oral SCC are treated with comprehensive neck dissection at the time of tumor resection, as salvage of patients who subsequently develop neck metastasis is poor. We propose that the +3q, -8p, +8q and +20 signature is suitable for stratifying patients for risk of metastasis prior to surgery, and so can guide surgical treatment for one of the most challenging treatment decisions - how to treat patients with clinically node-negative (N0) necks. Identification of patients with low risk for metastasis would spare them additional major surgery with its risks and morbidity, as well as reduce medical costs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5079. doi:1538-7445.AM2012-5079

Pancreatic ductal adenocarcinoma (PDA), the fourth-leading cause of cancer related death in the US, remains an incurable disease. Among the most promising targets for new treatments for PDA is the KRAS oncogene since mutations in this gene are found in about 90% of all cases. Because attempts to target the KRAS protein itself have failed so far, inhibiting pathways down-stream of KRAS in particular the MAPK and PI3K pathways, represent promising targets. We discovered that inhibition of MEK, a major effector of KRAS signaling, leads to feedback activation of the pro-survival PI3K/AKT pathway through an EGFR-mediated mechanism. In agreement with this finding, many PDA cell lines show synergistic growth inhibition and induction of apoptosis when treated with combinations of MEK and EGFR inhibitors or MEK with PI3K inhibitors. We profiled gene expression patterns in an extensive panel of human PDAC cell lines and identified two subtypes with epithelial and mesenchymal features, respectively. The vast majority of cell lines demonstrating synergism in response to combinations of MEK inhibitors (CI1040 or PD0325901) with EGFR inhibitors (erlotinib) were of epithelial sub-type. In contrast, none of the mesenchymal-type PDA lines showed synergistic effects with respect to apoptosis or cell growth inhibition. Predictor analysis using the cell line mRNA expression data revealed a 13-gene signature predictive of sensitivity to the drug combination. We hypothesize that these predictors will allow for the development of patient selection strategies for clinical application of combinations of MEK- and EGFR inhibitors in PDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A69.

9 Background: We developed a novel approach to isolate circulating tumor cells (CTCs) via immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) and examined copy number alterations in these cells. Methods: Magnetic beads coated with EpCAM mAb were added to blood to enrich for tumor cells. Enriched samples were then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. DNA from isolated tumor cells was subjected to whole genome amplification (WGA) and copy number analysis via array comparative genomic hybridization (aCGH). The assay was evaluated in CTCs from 5 MBC pts with matched archival primary tumors and later extended to an additional 176 MBC pts, 97 of which were successfully profiled. Results: Comparison of CTCs with matched archival primary tumors confirmed shared lineage with notable divergence. In addition, serial testing of CTCs confirmed reproducibility, and indicated genomic change over time. Genomic profiling of CTCs from 102 MBC pts revealed a wide range of copy number alterations including those previously reported in breast cancer. Comparison with a published aCGH dataset of primary breast tumors revealed similar frequencies of recurrent genomic copy number aberrations. Conclusions: It is feasible to isolate CTCs away from hematopoietic cells with high purity via IE/FACS and profile them via aCGH analysis following WGA. Our approach may be utilized to explore genomic events involved in cancer progression and to monitor therapeutic efficacy of targeted therapies in clinical trials in a relatively non-invasive manner. This work was supported by grants from the CALGB, BCRF, TBCRC (Avon, Komen), EDRN and U54.

6 Background: Metastatic CRPC undergoes significant genomic evolution compared to primary, localized disease. Multiple genetic mechanisms contribute to this evolution including changes in gene copy number as well as and changes in CpG island methylation. In this study, copy number and methylation status of metastatic CRPC tissue obtained at autopsy were assessed by high-resolution array comparative genomic hybridization (aCGH) and whole genome methylation microarray profiling. Methods: Array CGH was performed on DNA isolated from metastatic CRPC samples obtained from the University of Michigan Rapid Autopsy Program, using the Agilent Human Genome 244K CGH Microarray. A total of 15 samples comprising 7 metastatic liver implants and 8 soft tissue metastases were analyzed with DNA Analytics 4.0 software. Expression of genes identified as commonly amplified or deleted was assessed in a confirmatory fashion using existing Agilent 4×44 Expression Array data. The Illumina Infinium HumanMethylation27 BeadChip was used to determine whole genome methylation status. Results: A total of 79 amplified and 419 deleted genes common to >66% of samples were identified. There was significant correlation (p85% of samples, suggesting that inactivation of this tumor suppressor through multiple pathways is critical to tumor cell survival. Conclusions: This is the first known study examining both gene copy number and whole genome CpG island methylation status in metastatic CRPC. Integration of this data allows for identification of specific genes or pathways that confer a selective growth advantage to prostate cancer cells harboring those changes. No significant financial relationships to disclose.

2578 Background: CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells thereby serving as an immune checkpo...

The 5-year survival rate for oral squamous cell carcinoma (SCC) patients at 40% has not improved over the past 40 years. Since genomes of 75-80% of oral SCCs are characterized by regions of gains and losses, we investigated whether copy number aberrations could be used to address two important problems in oral cancer management - identification of (a) tumors with metastatic potential and (b) dysplastic lesions at risk for progression to cancer. Neck metastasis, the most significant clinical factor responsible for death from oral cancer reduces survival by 50%. To determine if copy number aberrations would distinguish tumors with metastatic potential, we carried out array comparative genomic hybridization (CGH) on 26 N0 and 38 N+ cases with at least 5-year follow up (diagnosed between 1997 and 2005). In order to reduce the multiple testing correction, regions of recurrent aberration were identified in an independent set of 89 oral SCCs (Snijders et al. 2005). A comparison of the frequency of aberrations affecting these regions in the N0 and N+ cases revealed no significant differences between the N0 and N+ cases after correction for multiple testing. We used a similar approach to determine if there are significant copy number differences between oral dysplasia and oral SCC. Most oral SCCs are preceded by precancerous lesions, characterized by varying degrees of dysplasia (graded from mild to severe). Transformation to oral SCC is associated with 16% of mild and 55% of moderate/severe dysplasia. Copy number profiles for 44 dysplasia samples comprised of approximately equal numbers of cases graded mild, moderate or severe were obtained by array CGH. Although fewer copy number changes were observed in dysplasia compared to SCC, gains of 3q, 8q and/or chromosome 20 were present at approximately equal frequencies in dysplasia and SCC. In addition, the rare narrow amplicons, characteristic of oral SCC (Snijders et al. 2005), were present in dysplasia. To identify copy number alterations distinguishing dysplasia and SCC, regions of recurrent aberration were defined in an independent set of oral SCCs (the 64 cases from the metastasis study described above). The frequency of these copy number alterations was compared between the dysplasia and the 89 SCC cases (Snijders et al. 2005). Gain of 7p, including EGFR in SCC was found to be significant after correction for multiple testing. This observation is consistent with previous reports of increased expression of EGFR with progression of dysplasia to cancer (Garnis et al., 1998). Since 20-25% of oral SCC harbor almost no copy number changes, suggesting that dysplasia lacking copy number changes may have potential to progress to SCC with chromosomally stable genomes, we suggest that measurement of EGFR expression and/or other genes on 7p may provide a more effective biomarker for dysplasia progression than 7p copy number. 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 2137.