Genomic and expression profiling reveal molecular heterogeneity of disseminated tumor cells in bone marrow of early breast cancer.

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.

Expanded Genomic Profiling of Circulating Tumor Cells in Metastatic Breast Cancer Patients to Assess Biomarker Status and Biology Over Time (CALGB 40502 and CALGB 40503, Alliance).

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.

Liver Metastasis and Treatment Outcome with Anti-PD-1 Monoclonal Antibody in Patients with Melanoma and NSCLC.

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.

Physical activity and prostate gene expression in men with low-risk prostate cancer.

PURPOSE: Vigorous physical activity after diagnosis of localized prostate cancer may reduce the risk of disease progression and prostate cancer-specific mortality. The molecular mechanisms by which physical activity may exert protective effects in the prostate remain unknown. METHODS: We examined the associations between self-reported physical activity and gene expression patterns in morphologically normal prostate tissue of 71 men with low-risk prostate cancer on active surveillance. Differential gene expression, gene set, and pathway analyses were conducted comparing dichotomous groups defined by type, intensity, and amount of physical activity reported. RESULTS: Cell cycling and DNA repair pathways were up-regulated in men who participated in ≥ 3 h/week vigorous activity compared with men who did not. In addition, canonical pathways involved in cell signaling and metabolism, the cellular effects of sildenafil (Viagra), and the Nrf2-mediated oxidative stress response were modulated in men who reported ≥ 3 h/week of vigorous activity. Differential expression analysis at the individual gene level revealed modest differences between men who performed vigorous activity for ≥ 3 h/week and those who did not. There were no differences in prostate gene expression in comparisons with exercise groupings that did not consider both duration and intensity of activity. CONCLUSIONS: Prostate gene expression and pathway analyses revealed sets of transcripts that may be modulated in normal prostate tissue by participating in ≥ 3 h/week of vigorous activity after diagnosis of low-risk prostate cancer. These findings suggest potential biological mechanisms by which vigorous activity may reduce risk of prostate cancer progression and warrant further study and validation.

Genome-wide copy number analysis of cerebrospinal fluid tumor cells and their corresponding archival primary tumors.

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.

Molecular profiling of tumor cells in cerebrospinal fluid and matched primary tumors from metastatic breast cancer patients with leptomeningeal carcinomatosis.

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.

Genomic profiling of isolated circulating tumor cells from metastatic breast cancer patients.

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.

Isolation and genomic analysis of circulating tumor cells from castration resistant metastatic prostate cancer.

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.

Gene expression and biological pathways in tissue of men with prostate cancer in a randomized clinical trial of lycopene and fish oil supplementation.

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.