Julie Ann Mayer

A Novel Platform for Detection of CK+ and CK− CTCs

UNLABELLED Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. SIGNIFICANCE Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.

Estrogen Induces c-myc Gene Expression via an Upstream Enhancer Activated by the Estrogen Receptor and the AP-1 Transcription Factor

c-myc oncogene is implicated in tumorigenesis of many cancers, including breast cancer. Although c-myc is a well-known estrogen-induced gene, its promoter has no estrogen-response element, and the underlying mechanism by which estrogen induces its expression remains obscure. Recent genome-wide studies by us and others suggested that distant elements may mediate estrogen induction of gene expression. In this study, we investigated the molecular mechanism by which estrogen induces c-myc expression with a focus on these distal elements. Estrogen rapidly induced c-myc expression in estrogen receptor (ER)-positive breast cancer cells. Although estrogen had little effect on c-myc proximal promoter activity, it did stimulate the activity of a luciferase reporter containing a distal 67-kb enhancer. Estrogen induction of this luciferase reporter was dependent upon both a half-estrogen response element and an activator protein 1 (AP-1) site within this enhancer, which are conserved across 11 different mammalian species. Small interfering RNA experiments and chromatin immunoprecipitation assays demonstrated the necessity of ER and AP-1 cross talk for estrogen to induce c-myc expression. TAM67, the AP-1 dominant negative, partially inhibited estrogen induction of c-myc expression and suppressed estrogen-induced cell cycle progression. Together, these results demonstrate a novel pathway of estrogen regulation of gene expression by cooperation between ER and AP-1 at the distal enhancer element and that AP-1 is involved in estrogen induction of the c-myc oncogene. These results solve the long-standing question in the field of endocrinology of how estrogen induces c-myc expression.

FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform.

Determination of HER2 status in breast cancer patients is considered standard practice for therapy selection. However, tumor biopsy in patients with recurrent and/or metastatic disease is not always feasible. Thus, circulating tumor cells (CTCs) are an alternative source of tumor cells for analysis of HER2. An antibody cocktail for recovery of variable, high- and low-, EpCAM-expressing tumor cells was developed based on FACS evaluation and then verified by CTC enumeration (based on CK and CD45 staining) with comparison to EpCAM-only and with CellSearch® (n=19). HER2 fluorescence in situ hybridization (FISH) on all (CK+ and CK-) captured cells was compared to HER2 status on the primary tumors (n=54) of patients with late stage metastatic/recurrent breast cancer. Capture of low EpCAM-expressing tumor cells increased from 27% to 76% when using the cocktail versus EpCAM alone, respectively. Overall, CTC detection with the OncoCEE™ platform was better compared to CellSearch® (68% vs. 89%, respectively), and a 93% concordance in HER2 status was observed. HER2 FISH analysis of CK+ and CK- CTCs is feasible using the CEE™ platform. Although larger clinical studies are warranted, the results demonstrate adequate sensitivity and specificity as needed for incorporation into laboratory testing.

Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer

Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5–10 mL) were collected prospectively from patients with clinical stages I–IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK−/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45−/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2− primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2− primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.

The rearranged during transfection/papillary thyroid carcinoma tyrosine kinase is an estrogen-dependent gene required for the growth of estrogen receptor positive breast cancer cells

The rearranged during transfection/papillary thyroid carcinoma (RET/PTC) tyrosine kinase is an oncogene implicated in the tumorigenesis of thyroid cancer. Recent studies by us and others have shown that RET/PTC kinase expression is induced by estrogen in breast cancer cells. Due to the critical involvement of estrogen-regulated genes in the pathogenesis of breast cancer, we investigated the expression, regulation, and function of RET/PTC kinase in breast cancer cells. We found that RET/PTC kinase expression correlates with estrogen receptor (ER) expression in breast cancer cells and tumor specimens, and that RET/PTC kinase expression is associated with a poor prognosis in ER-positive breast cancer patients. We found that estrogen rapidly induces RET/PTC kinase expression in an ER-dependent manner in breast cancer cells and that this induction is through a transcriptional regulatory mechanism. Using reporter assays, small interfering RNA (siRNA) assays, and chromatin immunoprecipitation (ChIP) assays, we demonstrated the necessity of crosstalk between ER and the forkhead box A1 (FOXA1) transcription factor in regulating RET/PTC kinase expression. In functional studies, increased expression of RET/PTC kinase induced by estrogen stimulation resulted in elevated phosphorylation of multiple downstream kinase signaling pathways. Conversely, knockdown of RET/PTC expression was associated with the inhibition of these same kinase signaling pathways, and, in fact, decreased the stimulatory effect of estrogen on the proliferation of ER-positive breast cancer cells. These results demonstrate a novel pathway of ER and FOXA1 transcription factor crosstalk in regulating RET/PTC kinase expression, and demonstrate that RET/PTC kinase is a critical regulator for the proliferation of ER-positive breast cancer cells. Taken together, our study suggests that RET/PTC kinase may serve as a novel prognostic biomarker and therapeutic target for prevention and treatment of ER-positive breast cancer.

SLC22A5/OCTN2 expression in breast cancer is induced by estrogen via a novel intronic estrogen-response element (ERE)

Estrogen signaling is a critical pathway that plays a key role in the pathogenesis of breast cancer. In a previous transcriptional profiling study, we identified a novel panel of estrogen-induced genes in breast cancer. One of these genes is solute carrier family 22 member 5 (SLC22A5), which encodes a polyspecific organic cation transporter (also called OCTN2). In this study, we found that estrogen stimulates SLC22A5 expression robustly in an estrogen receptor (ER)-dependent manner and that SLC22A5 expression is associated with ER status in breast cancer cell lines and tissue specimens. Although the SLC22A5 proximal promoter is not responsive to estrogen, a downstream intronic enhancer confers estrogen inducibility. This intronic enhancer contains a newly identified estrogen-responsive element (ERE) (GGTCA-CTG-TGACT) and other transcription factor binding sites, such as a half ERE and a nuclear receptor related 1 (NR4A2/Nurr1) site. Estrogen induction of the luciferase reporter was dependent upon both the ERE and the NR4A2 site within the intronic enhancer. Small interfering RNA against either ER or Nurr1 inhibited estrogen induction of SLC22A5 expression, and chromatin immunoprecipitation assays confirmed the recruitment of both ER and Nurr1 to this enhancer. In functional assays, knockdown of SLC22A5 inhibited l-carnitine intake, resulted in lipid droplet accumulation, and suppressed the proliferation of breast cancer cells. These results demonstrate that SLC22A5 is an estrogen-dependent gene regulated via a newly identified intronic ERE. Since SLC22A5 is a critical regulator of carnitine homeostasis, lipid metabolism, and cell proliferation, SLC22A5 may serve as a potential therapeutic target for breast cancer in the future.

Abstract 3734: Development of a nanostring copy number assay for a customized 55 gene panel using challenging formalin-fixed paraffin-embedded (ffpe) tumor samples

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Gene amplification/deletion is a common tactic employed by tumor cells to proliferate uncontrollably. Multiple methods have been developed to determine gene copy numbers, such as FISH, qPCR, and, more recently, Next Generation Sequencing (NGS). However, these methods have limitations: FISH and NGS are very labor intensive; and FISH and qPCR can detect only a limited number of targets. Therefore, it is imperative to develop methods that are more user-friendly and have increased multiplexing capabilities to detect copy number variations (CNV). Using a novel digital barcode technology, NanoString Technologies (Seattle, WA) has developed the nCounter® technology to meet these needs. This new technology will be especially valuable after the recent FDA approval of the ProsignaTM assay, a breast cancer gene signature assay for subtype classification to help guide treatment decisions. In the BioPharma group at Genoptix, Inc., Carlsbad, CA, we have started to assess the nCounter technology for copy number determinations in a CLIA laboratory setting. Methods: A cancer gene CNV panel composed of 55 genes was custom designed and evaluated. The CNV results of a subset of genes generated from our gene panel using the nCounter technology were compared with the results generated from qPCR, digital droplet PCR (ddPCR), FISH, publications, and NGS. Furthermore, assay robustness was tested by using different amounts and quality of input FFPE DNA. An initial comparison on the CNVs obtained from genomic DNA digested by Alu1 enzyme or sonicated by Bioruptor® Pico (Diagenode Inc, Denville, NJ). Finally, the effects of different DNA isolation methods on copy number determinations were also evaluated. Results: Overall, the copy numbers generated using nCounter technology were concordant with results from the abovementioned methods as analyzed by comparing selected genes with known CNVs. A wide range of FFPE DNA input (200ng-4ug) could be tolerated by the assay. Based on a limited number of samples, DNA isolation methods, such as Qiagen, Promega Maxwell® CSC, or Roche Cobas®, have minimal effect on copy numbers. Conclusions: With the short hands-on time, relative high level of automation, and no amplification requirements, nCounter technology is able to generate CNVs in good agreement with other established CNV methods. This new 55 cancer gene CNV assay may be useful in clinical trials to assist in patient stratification and care. Citation Format: Tingdong Tang, Wenge Shi, Loretta Hipolito, Julie Mayer, Jelveh Lameh, Shabnam Tangri, Reinhold Pollner. Development of a nanostring copy number assay for a customized 55 gene panel using challenging formalin-fixed paraffin-embedded (ffpe) tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3734. doi:10.1158/1538-7445.AM2014-3734

Abstract 4568: Estrogen receptor and progesterone receptor immunocytochemistry staining in circulating tumor cells as compared to primary tumor or metastatic biopsy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Hormone receptor (estrogen receptor (ER) and progesterone receptor (PR)) status in all breast cancer patients is recommended by immunohistochemistry (IHC) and is considered standard practice for selection of treatment options. However, the analytical sensitivity of IHC in detecting low levels of ER/PR is often poor and likely due to methodological variation. Because biopsy is not often feasible in all patients presenting with recurrent and/or metastatic breast disease, circulating tumor cells (CTCs) offer an attractive alternative source of tumor tissue for determining ER/PR status and can be monitored more readily to enable a more effective course of treatment. Experimental Design: Twenty ml of peripheral blood was collected prospectively from 14 patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic OncoCEETM platform. A cocktail of antibodies was utilized for CTC capture and detection with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on the cells captured within the microchannels and compared to IHC performed on the primary tumor or metastatic biopsy. Results: CK+/CD45-/DAPI+ cells were located and assessed for ER/PR immunocytochemistry. Among the 14 cases a high concordance (12/14; 86%) in ER/PR status between primary tumor/metastatic biopsy and CTCs was observed. Two cases were found to be discordant where one was positive by IHC and negative on the CTCs and the other was negative on by IHC on the primary tumor/metastatic biopsy and positive on the CTCs. However, both cases that were discordant had low numbers of CTCs detected. Conclusions: There is significant heterogeneity between ER/PR protein expression in CTCs and primary tumor/metastatic biopsy and this status may change over time due to therapy. ER/PR ICC on CTCs from peripheral blood using the OncoCEETM platform is shown to be feasible with high concordance (86%) in ER/PR status between primary tumor/metastatic biopsy (by IHC) and CTCs (by ICC). The significance of heterogeneity at the ER/PR protein level in CTCs ascertaining to the prognosis and predictive response to anti-estrogen therapy needs further evaluation in larger prospective clinical trials. 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 4568. doi:1538-7445.AM2012-4568

Abstract P2-01-10: Immunocytochemistry staining for estrogen and progesterone receptor in circulating tumor cells: Concordance between primary and metastatic tumors.

Introduction: Estrogen (ER) and progesterone receptor (PR)) status is recommended by immunohistochemistry (IHC) and is considered standard practice for selection of treatment options in all breast cancer patients. Because biopsy is not often feasible in every patient presenting with recurrent and/or metastatic disease, circulating tumor cells (CTCs) offer an attractive alternative source of tumor tissue for determining ER/PR status. In addition, CTCs enable monitoring for more effective course of treatment. Experimental Design: Twenty ml of peripheral blood was collected prospectively from 34 patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic OncoCEE platform. A cocktail of antibodies was utilized for CTC capture, and detection was accomplished with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on CK+/CD45- CTCs captured directly within the microchannel and then compared to IHC performed on the primary and/or metastatic tumor. Results: CK+/CD45−/DAPI+ cells were detected in 22 of 34 (65%) patients with late stage breast cancer and assessed for ER/PR immunocytochemistry. Among the 22 cases with one or more CTCs, a concordance of 75% (15/20) and 90% (9/10) in ER/PR status between primary and metastatic tumor was observed, respectively. An overall concordance of 86% (19/22) was achieved. Five cases were discordant based on primary tissue alone; however, two of these cases are concordant when compared to the metastatic biopsy. Thus, only three cases were found to be discordant: all three were positive by IHC on the primary and/or metastatic tumor but negative by CTCs and all three had relatively low numbers of CTCs detected. Conclusions: There is significant heterogeneity of ER/PR protein expression in CTCs and primary/metastatic tumor biopsy. In addition, hormonal status may change over time due to therapy. ER/PR ICC on CTCs using the OncoCEE platform is shown to be feasible, with high concordance (86%) as compared to primary and/or metastatic biopsy (by IHC). The significance of heterogeneity at the ER/PR protein level in CTCs related to the prognosis and predictive response to anti-estrogen therapy needs further evaluation in larger prospective clinical trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-10.

Abstract LB-310: FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose : Determination of HER2 status by immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) in patients with breast cancer is considered standard practice for selection of treatment options. Though patients presenting with recurrent and/or metastatic disease are often re-evaluated for HER2, biopsy is not often feasible. Thus, circulating tumor cells (CTCs) are an attractive alternative source of tumor tissue for determining HER2 status to enable a more effective course of treatment. Experimental Design : Twenty to thirty ml of peripheral blood was collected prospectively from 54 patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic CEE™ platform. CTC capture was achieved using a cocktail of capture antibodies, followed by detection with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. HER2 amplification was subsequently assessed by FISH on captured CK+/CD45− and CK−/CD45− cells. Results : CK+/CD45− cells were detected in 43 of 54 cases (80%). Among the 43 cases in which CK+ cells were detected, high concordance (93%) in HER2 status between primary tumor (by IHC and FISH) and CTCs (by FISH) was observed. An overall sensitivity of 95% and a specificity of 92% were obtained using the OncoCEE-BR™ assay. Conclusions : Recovery of CTCs from peripheral blood using the CEE™ platform is shown to be efficient and suitable for FISH-based testing. In addition, HER2 FISH on recovered CTCs is proven to be sensitive and accurate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-310. doi:10.1158/1538-7445.AM2011-LB-310