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Quantitative CD8+ Counts in patients with Liver and skin metastases.

Univariate Analysis of Pretreatment prognostic factors in the Discovery cohort.

Supplementary Figure 1. Development, optimization and performance of the gene expression assay. Supplementary Figure 2. Evaluation of assay performance using cancer cell line spike-in models. Supplementary Figure 3. Sample processing and quality control measures Supplementary Figure 4. Expression analysis of CTCs and matched normal blood samples. Supplementary Figure 5. Subtype and proliferation status. Supplementary Figure 6. Serial analysis. Supplementary Figure 7. Clustering analysis of copy number data. Supplementary Figure 8. ESR1 and ERBB2 status in CTCs and patient outcome Supplementary Figure 9. CTC molecular subtype and patient outcome.

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.

PDF file - 965K, FIG S1: Distribution of MAD estimates across all 102 circulating tumor cell samples FIG S2: Visualization of IE/FACS-isolated cells via hematoxylin/eosin staining; FIG S3: Visualization of IE/FACS-isolated cells via fluorescence in-situ hybridization (FISH) analysis; FIG S4: aCGH analysis of spiked BT474 cells at the single cell level; FIG S5: aCGH analysis of spiked MCF7 cells; FIG S6: Study flow chart; FIG S7: Sorting replication in Patient #303; FIG S8: Comparison of circulating tumor cells and matched primary tumor

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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.

We explored the association between liver metastases, tumor CD8+ T-cell count, and response in patients with melanoma or lung cancer treated with the anti-PD-1 antibody, pembrolizumab. The melanoma discovery cohort was drawn from the phase I Keynote 001 trial, whereas the melanoma validation cohort was drawn from Keynote 002, 006, and EAP trials and the non–small cell lung cancer (NSCLC) cohort from Keynote 001. Liver metastasis was associated with reduced response and shortened progression-free survival [PFS; objective response rate (ORR), 30.6%; median PFS, 5.1 months] compared with patients without liver metastasis (ORR, 56.3%; median PFS, 20.1 months) P ≤ 0.0001, and confirmed in the validation cohort (P = 0.0006). The presence of liver metastasis significantly increased the likelihood of progression (OR, 1.852; P < 0.0001). In a subset of biopsied patients (n = 62), liver metastasis was associated with reduced CD8+ T-cell density at the invasive tumor margin (liver metastasis+ group, n = 547 ± 164.8; liver metastasis− group, n = 1,441 ± 250.7; P < 0.016). A reduced response rate and shortened PFS was also observed in NSCLC patients with liver metastasis [median PFS, 1.8 months; 95% confidence interval (CI), 1.4–2.0], compared with those without liver metastasis (n = 119, median PFS, 4.0 months; 95% CI, 2.1–5.1), P = 0.0094. Thus, liver metastatic patients with melanoma or NSCLC that had been treated with pembrolizumab were associated with reduced responses and PFS, and liver metastases were associated with reduced marginal CD8+ T-cell infiltration, providing a potential mechanism for this outcome. Cancer Immunol Res; 5(5); 417–24. ©2017 AACR.

BACKGROUND Observational studies suggest an inverse association between selenium and risk of prostate cancer. However, randomized controlled trials of selenium supplementation have reported conflicting results. Thus, we examined plasma selenium and selenium-related genes in relation to risk of high-grade prostate cancer and prostate cancer recurrence among men initially diagnosed with non-metastatic disease. METHODS We measured plasma selenium and genotyped 73 single nucleotide polymorphisms in TXNRD1, TXNRD2, GPX1, GPX3, GPX4, SEP15, SEPP1, SELENBP1, OGG1, and CAT among 568 men with non-metastatic prostate cancer who underwent radical prostatectomy. We examined associations between plasma selenium, genotypes, and risk of high-grade prostate cancer (Gleason grade ≥8 or 7 with primary score ≥4; n = 111) using logistic regression, and risk of prostate cancer recurrence (61 events; 3.8 y median follow-up) using Cox proportional hazards regression. RESULTS Plasma selenium was not associated with risk of high-grade prostate cancer or prostate cancer recurrence. Less common alleles of rs11913319 in TXNRD2 and rs125701 in OGG1 were associated with an increased risk of high-grade prostate cancer. We observed associations between the risk of prostate cancer recurrence and multiple SNPs in TXNRD1, TXNRD2, GPX3, and SEP15. These associations were no longer statistically significant after adjustment for multiple comparisons. CONCLUSIONS Among men with non-metastatic prostate cancer, there is suggestive evidence that genetic variation in selenoproteins and related antioxidant enzymes may be associated with risk of high-grade disease at diagnosis and prostate cancer recurrence. Prostate 75:60–69, 2015. © 2014 Wiley Periodicals, Inc.

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.

BACKGROUND Observational studies suggest an inverse association between vitamin E and risk of prostate cancer, particularly aggressive tumors. However, three large randomized controlled trials have reported conflicting results. Thus, we examined circulating vitamin E and vitamin E-related genes in relation to risk of high-grade prostate cancer and prostate cancer recurrence among men initially diagnosed with clinically organ-confined disease. METHODS We measured circulating α- and γ-tocopherol and genotyped 30 SNPs in SOD1, SOD2, SOD3, TTPA, and SEC14L2 among 573 men with organ-confined prostate cancer who underwent radical prostatectomy. We examined associations between circulating vitamin E, genotypes, and risk of high-grade prostate cancer (Gleason grade ≥ 8 or 7 with primary score ≥ 4; n = 117) using logistic regression, and risk of recurrence (56 events; 3.7 years median follow-up) using Cox proportional hazards regression. RESULTS Circulating γ-tocopherol was associated with an increased risk of high-grade prostate cancer (Q4 v. Q1 odds ratio [OR] = 1.87; 95% confidence intervals [CI]: 0.97–3.58; Ptrend = 0.02). The less common allele in SOD3 rs699473 was associated with an increased risk of high-grade disease (T > C: OR = 1.40, 95% CI: 1.04–1.89). Two independent SNPs in SOD1 were inversely associated with prostate cancer recurrence in additive models (rs17884057 hazard ratio [HR] = 0.49, 95%CI: 0.25–0.96; rs9967983 HR = 0.62, 95% CI: 0.40–0.95). CONCLUSIONS Among men with clinically organ-confined prostate cancer, genetic variation in SOD may be associated with risk of high-grade disease at diagnosis and disease recurrence. Circulating γ-tocopherol levels may also be associated with an increased risk of high-grade disease at diagnosis. Prostate 73:1786–1795, 2013. © 2013 Wiley Periodicals, Inc.

Background: The paucity of information regarding the molecular nature of CTCs can be largely attributed to the difficulty of isolating these rare cells. As such, efforts on RNA profiling of CTCs have been limited to studies using immuno-enriched samples containing a high background of leukocytes. Methods: We developed a two-step process: immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) to isolate CTCs away from leukocytes and performed expression analysis on a limited panel of genes. Magnetic beads coated with EpCAM mAb were used to enrich for tumor cells. Enriched samples were then subjected to FACS using fluorescently labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. CTCs and matched leukocytes were isolated from 67 sequential patients with MBC. Total RNA from isolated 20–50 cells was reverse-transcribed into cDNA. Sixty four (64) CTC-related genes from previous published reports were chosen for expression analysis. cDNA of these genes were pre-amplified and analyzed in triplicate via Taqman®-based RT-PCR in a low density array format. Statistical analysis of gene expression data was performed using Realtime Statminer®. Results: Unsupervised hierarchical clustering analysis showed that CTCs clustered away from the leukocytes. Differential gene expression analysis revealed the up-regulation of several genes including EPCAM, MUC1, and KRT19 in CTCs (adjusted p Discussion: We demonstrate the feasibility of isolation and gene expression analysis of CTCs. Molecular profiling confirmed that isolated CTCs were highly pure with little or no evidence of leukocyte contamination. This approach can serve as a non-invasive source of tumor tissue for further molecular characterization of CTCs and for monitoring of therapeutic efficacy of targeted therapies in clinical trials. This study was supported by CALGB and BCRF. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-17.

A debilitating complication of breast cancer is the metastatic spread of tumor cells to the leptomeninges or cerebrospinal fluid (CSF). Patients diagnosed with this aggressive clinical syndrome, known as leptomeningeal carcinomatosis, have very poor prognosis. Despite improvements in detecting cerebrospinal fluid tumor cells (CSFTCs), information regarding their molecular biology is extremely limited. In our recent work, we utilized a protocol previously used for circulating tumor cell isolation to purify tumor cells from the CSF. We then performed genomic characterization of CSFTCs as well as archival tumors from the same patient. Here, we describe the microarray data and quality controls associated with our study published in the Cancer Research journal in 2013 [1]. We also provide an R script containing code for quality control of microarray data and assessment of copy number calls. The microarray data has been deposited into Gene Expression Omnibus under accession # GSE46068.

Background: Clinical studies have demonstrated the prognostic and predictive value of the number of CTCs in metastatic breast cancer. However, it is the molecular characterization of CTCs that offers insight into the biology of these tumor cells in the context of personalized treatment. Methods: Ten to twenty mLs of blood from 32 metastatic breast cancer patients with poor prognosis (≥5 CTCs in 7.5mLs blood) were subjected to immunomagnetic enrichment. Pools of ∼20 CTCs [EpCAM+, CD45-, nucleated] were isolated via fluorescence activated cell sorting, FACS. Genomic DNA was subjected to whole genome amplification followed by array comparative genomic hybridization (CGH) analysis. Serial genomic profiling was performed in three patients. Genomic profiles from archival primary tumor available from 4 patients were compared to matched CTCs. Copy number analysis was performed using the Nexus® Copy Number. Genomic alterations with p ≥0.05 were considered significant. Results: Genomic profiling of CTCs revealed significant recurrent (≥35%) genomic alterations including gains in 8q12.1, 8q24 and losses in 1p36, 4p16, 5q21, 10q22, 11q24-25, and 13q34. Good concordance of CGH profiles obtained from CTCs isolated from serial blood samples attested to the reproducibility of the assay. Furthermore, comparisons of CTCs with matched archival primary tumors confirmed shared lineage with some divergence. Conclusions: Copy number analysis revealed common genomic alterations in CTCs as well as shared aberrations with matched primary tumor. In addition, we demonstrated the feasibility of serial genomic profiling of CTCs. Future work will determine associations between genomic aberrations in CTCs and clinicopathological parameters. Uncovering genomic alterations in CTCs may lead to the discovery of therapeutic biomarkers to specifically target these cells in breast cancer patients who respond poorly to current aggressive therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-10.

Background Nutritional factors are associated with reduced risk of prostate cancer progression, yet mechanisms remain unclear. We examined the effects of lycopene and fish oil supplements versus placebo on the normal prostate microenvironment, among men pursuing active surveillance for low-burden prostate cancer. We hypothesized that lycopene or fish oil supplements would down-regulate insulin-like growth factor-1 (IGF-1) and cyclooxygenase 2 (COX-2) gene expression, respectively, reflecting putative proliferation (IGF-1) and inflammatory (COX-2) pathways relevant to carcinogenesis. Methods We conducted a 3-month randomized, double-blinded, clinical trial comparing prostate tissue gene expression profiles (assessed by qRT–PCR) among men with favorable-risk prostate cancer receiving either 30 mg/day lycopene, 3 g/day fish oil (including 1,098 mg eicosapentaenoic and 549 mg docosahexaenoic fatty acids) or placebo. Results Among 69 men (22 assigned to lycopene, 21 to fish, and 26 to placebo), there was no difference in the change from baseline to the 3 months in IGF-1 expression level between the placebo and lycopene arms (p = 0.93) nor in COX-2 expression between the placebo and fish arms (p = 0.99). Conclusion Compared to placebo, 3-month intervention with lycopene or fish oil did not significantly change IGF-1 and COX-2 gene expression in the normal prostate microenvironment in men with low-burden prostate cancer. Further analysis of global gene expression profiles may shed light on the bioactivity and relevance of these nutrients in prostate cancer.

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.

189 Background: Physical activity (PA), in particular longer duration or higher intensity, may reduce the risk of PCa progression and PCa-specific mortality in men diagnosed with clinically localized PCa. However, the molecular mechanism(s) by which PA exerts its protective effect in the prostate remains unknown. We examined the correlation of PA and gene expression patterns in men with low risk prostate cancer who elected to undergo active surveillance. Methods: Morphologically normal prostate tissue was obtained from men who subsequently participated in a clinical trial focused on nutritional supplements (previously published microarray dataset #GSE27140). Of the original sample (n=84), 70 completed a brief PA questionnaire and were dichotomized based usual PA [e.g. any vigorous PA (yes/no), 3+ h/wk vigorous PA (yes/no)]. Differential expression and pathway (gene set) analyses between groups were performed using Significance Analysis of Microarrays. Genes and gene sets with a false discovery rate ≤0.10 and 0.20 were considered significant, respectively. Results: Gene expression analysis detected 184 significant genes that were differentially expressed between men who performed vigorous PA for 3+ h/wk (n=23) and those who did not (n= 47). Up-regulated genes included the known tumor suppressors, BRCA1 and BRCA2. Furthermore, pathway analysis revealed that cell cycle and DNA repair pathways were positively modulated in men who participated in 3+h/wk vigorous PA vs. not. Consistent with the data on vigorous PA and clinical outcomes in men with PCa, the duration of vigorous PA was important; there were no significant genes detected when comparing men who participated in any vigorous PA to men who did none. Conclusions: Prostate gene expression and pathway analyses revealed candidate genes and in vivo pathways that may be modulated by participating in 3+ h/wk of vigorous PA. These data provide mechanistic insight into how 3+ h/wk of vigorous PA may offer PCa-specific benefits. Furthermore, understanding the molecular mechanisms by which such PA affects normal prostate gene expression may aid the development of strategies to prevent or delay PCa progression.

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.

Background The number of circulating tumor cells (CTCs) in metastatic prostate cancer patients provides prognostic and predictive information. However, it is the molecular characterization of CTCs that offers insight into the biology of these tumor cells in the context of personalized treatment. Methods We developed a novel approach to isolate CTCs away from hematopoietic cells with high purity, enabling genomic analysis of these cells. The isolation protocol involves immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS). To evaluate the feasibility of isolation of CTCs by IE/FACS and downstream genomic profiling, we conducted a pilot study in patients with metastatic castration resistant prostate cancer (CRPC). Twenty (20) sequential CRPC patients were assayed using CellSearch™. Twelve (12) patients positive for CTCs were subjected to immunomagnetic enrichment and fluorescence activated cell sorting (IE/FACS) to isolate CTCs. Genomic DNA of CTCs was subjected to whole genome amplification (WGA) followed by gene copy number analysis via array comparative genomic hybridization (aCGH). Results CTCs from nine (9) patients successfully profiled were observed to have multiple copy number aberrations including those previously reported in primary prostate tumors such as gains in 8q and losses in 8p. High-level copy number gains at the androgen receptor (AR) locus were observed in 7 (78%) cases. Comparison of genomic profiles between CTCs and archival primary tumors from the same patients revealed common lineage. However, high-level copy number gains in the AR locus were observed in CTCs, but not in the matched archival primary tumors. Conclusions We developed a new approach to isolate prostate CTCs without significant leukocyte admixture, and to subject them to genome-wide copy number analysis. Our assay may be utilized to explore genomic events involved in cancer progression, e.g. development of castration resistance and to monitor therapeutic efficacy of targeted therapies in clinical trials in a relatively non-invasive manner.

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

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

Background: Molecular studies of EpCAM-positive cells found in the blood of cancer patients have been limited to immuno-enriched samples containing a high background of leukocytes. We developed a process to isolate rare EpCAM-positive cells and subjected them to DNA and RNA profiling. Methods: Blood was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). Duplicate samples of 20 cells were isolated from the same enriched blood from 53 MBC patients and then subjected to DNA and RNA profiling in parallel. For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays. For non-tumor controls, RNA profiling was performed on matched leukocytes (CD45+) isolated from the same enriched blood samples from 44 of the 53 patients. Results: Differential gene expression analysis between EpCAM-positive cells and matched leukocytes confirmed the up-regulation of EPCAM and other genes including MUC1 and KRT19 (adjusted p Discussion: We demonstrate the feasibility of isolation and molecular analysis of highly pure EpCAM-positive cells in the blood. Our results strongly suggest that epithelial cells captured from blood of MBC patients are malignant in nature and are therefore circulating tumor cells. This approach can serve as a non-invasive source of highly pure metastatic tumor tissue for further molecular characterization. This study was supported by CALGB and BCRF. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-05.

33 Background: Laboratory and genomic data suggest that the antioxidant defense system is important in prostate carcinogenesis, and that this relation may vary by plasma antioxidant status. Methods: Among 567 radical prostatectomy (RP) patients with non-metastatic prostate cancer (CaP), we examined genetic variants in SOD1(5 SNPs), SOD2(6), SOD3(6), TXRND1(5), TXRND2(21), GPX1(3), GPX3(8), GPX4(5), CAT(9), XRCC1(2), SELENBP1(5), SEP15(4), SEPP1(4), SEC14L2(8), and TTPA(4) and risk of high pathologic Gleason sum (8+ or 7 with primary 4+) using logistic regression, and CaP recurrence (PSA rise, second treatment, metastasis) using Cox proportional hazards regression. We also examined whether the genetic variants interacted with plasma selenium, alpha-tocopherol, or gamma-tocopherol in relation to risk of high grade CaP. Results: 21% (n = 117) of men had high grade CaP, and 47 recurrences occurred (median follow-up of all participants = 3 y). Men with 1+ of the less common allele (CT or TT) in rs4880 (SOD2) had a 49% reduced rate of recurrence (hazard ratio (HR): 0.51; 95% confidence interval (CI): 0.28, 0.92), independent of grade, stage, and PSA. In contrast, men with 1+ of the less common allele in 3 of the TXRND1, 4 of the TXRND2, and 1 of the SEP15 SNPs had 2-fold increased rate of recurrence (HRs: 1.93-2.95; p-values: 0.002-0.04), and men with 2 of the less common allele in 2 of the GPX3 SNPs had 3-fold increased rate of recurrence (p-values from additive models: Conclusions: Genetic variants in antioxidant genes may be associated with CaP recurrence after RP, and may interact with plasma selenium and alpha-tocopherol in relation to risk of high grade CaP.

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.

10515 Background: To elucidate candidate genes and pathways associated with poor response, we retrospectively analyzed gene expression profiles in serial biopsies from women with locally advanced breast cancer who failed to respond to anthracycline-based chemo followed by taxane in the I-SPY TRIAL (CALGB150007/150012/ACRIN 6657). Methods: Of the 221 patients who completed neoadjuvant chemotherapy, 215 patients had surgery with 73% not achieving a pathologic complete response (pathCR). In these patients, cDNA microarray expression profiles from pretreatment biopsy (T1) were compared to those biopsy specimens obtained 24-72 hours after initiation of treatment (T2) or in tumors surgically removed after chemotherapy (TS). Paired expression data for T1vsT2 and T1vsTS were available for 29 and 39 patients with no pathCR, respectively. Paired differential expression analyses were performed via significance analysis of microarrays (SAM) and differentially expressed genes were subjected to ingenuity pathway analys...