Sreeraj G. Pillai

Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration.

Purpose Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays. Methods BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression. Results Filtering an average of 14×106 nucleated BM cells yielded approximately 17–21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84–92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM. Conclusions We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.

Identifying biomarkers of breast cancer micrometastatic disease in bone marrow using a patient-derived xenograft mouse model

BackgroundDisseminated tumor cells (DTCs) found in the bone marrow (BM) of patients with breast cancer portend a poor prognosis and are thought to be intermediaries in the metastatic process. To assess the clinical relevance of a mouse model for identifying possible prognostic and predictive biomarkers of these cells, we have employed patient-derived xenografts (PDX) for propagating and molecularly profiling human DTCs.MethodsPreviously developed mouse xenografts from five breast cancer patients were further passaged by implantation into NOD/SCID mouse mammary fat pads. BM was collected from long bones at early, serial passages and analyzed for human-specific gene expression by qRT-PCR as a surrogate biomarker for the detection of DTCs. Microarray-based gene expression analyses were performed to compare expression profiles between primary xenografts, solid metastasis, and populations of BM DTCs. Differential patterns of gene expression were then compared to previously generated microarray data from primary human BM aspirates from patients with breast cancer and healthy volunteers.ResultsHuman-specific gene expression of SNAI1, GSC, FOXC2, KRT19, and STAM2, presumably originating from DTCs, was detected in the BM of all xenograft mice that also developed metastatic tumors. Human-specific gene expression was undetectable in the BM of those xenograft lines with no evidence of distant metastases and in non-transplanted control mice. Comparative gene expression analysis of BM DTCs versus the primary tumor of one mouse line identified multiple gene transcripts associated with epithelial-mesenchymal transition, aggressive clinical phenotype, and metastatic disease development. Sixteen of the PDX BM associated genes also demonstrated a statistically significant difference in expression in the BM of healthy volunteers versus the BM of breast cancer patients with distant metastatic disease.ConclusionUnique and reproducible patterns of differential gene expression can be identified that presumably originate from BM DTCs in mouse PDX lines. Several of these identified genes are also detected in the BM of patients with breast cancer who develop early metastases, which suggests that they may be clinically relevant biomarkers. The PDX model may also provide a clinically relevant system for analyzing and targeting these intermediaries of metastases.

Abstract P5-04-06: Analysis of STEAP1 expression as a therapeutic target in breast cancer

Objective: The six transmembrane epithelial antigen of the prostate (STEAP1) is predominantly overexpressed in human prostate cancer. STEAP1 was first identified as a prostate-specific cell-surface antigen and found to be up-regulated in various cancers including lung, bladder, colon, and ovarian with little expression in normal tissue. An anti-STEAP1 monoclonal antibody linked to an antimitotic agent is currently in Phase I clinical trials for prostate cancer patients. Microarray data from our lab suggested that STEAP1 is also highly expressed in human breast cancers and bone marrow disseminated tumor cells. In this study we evaluate expression STEAP1 in primary tumors, and bone marrow (BM) from breast cancer patients. Experimental procedures: RNA was isolated from primary tumor, non-malignant breast tissue and bone marrow (BM) from stage II and III breast cancer patients, healthy volunteers, breast cancer cell lines and BM from patient derived xenographs (PDX). Disseminated tumor cells (DTCs) from patient BM were enriched by microfiltration and analyzed by RNA-in situ hybridization (ISH). STEAP1 RNA expression was analyzed by Nanostring nCounter and qRT-PCR using human specific probes. STEAP1 immunohistochemical (IHC) staining of human tissue was performed using standard protocols. Knockdown of steap1 expression was accomplished using a lentiviral system. Results: STEAP1 mRNA was up-regulated in 77% of tumors (28/36) compared to the corresponding normal tissue. STEAP1 protein was expressed in 100% of tumors (8/8) and was absent in non-malignant breast tissue (7/7) by IHC staining. STEAP1 mRNA was not expressed normal BM, but was detected in 8% (6/74) of BM from patients with early stage breast cancer. STEAP1 expression in the BM was associated with triple negative disease (3/6) and recurrent disease development (4/6, p=.028). STEAP1 expression was observed in individual DTCs isolated from patients BM, while no expression was observed in normal BM. In a PDX model of breast cancer, STEAP1 expression in BM was only observed in mouse who developed metastatic disease associated (7/10, p=.004). Knockdown of STEAP1 in the breast cancer cell line MDA-MB231 cells inhibited cell growth by 80-90%. Conclusion: STEAP1 is expressed in human breast tumors and disseminated tumor cells found in the bone marrow of breast cancer patients. Expression of STEAP1 in the BM is significantly associated with the development of metastatic disease in patients as well as in a mouse model of breast cancer. Our data indicate that STEAP1 could serve as a therapeutic target for the treatment of minimal residual disease in breast cancer. Citation Format: Aft R, Mudalagiriyappa C, Pillai S, Watson M. Analysis of STEAP1 expression as a therapeutic target in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-06.

Abstract 5258: Expression analysis of STEAP1 in breast cancer patients as therapeutic target

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objective: The six transmembrane epithelial antigen of the prostate (STEAP1) is predominantly overexpressed in human prostate cancer. STEAP1 was first identified as a prostate-specific cell-surface antigen and found to be up-regulated in multiple cancer cell lines and in various cancers, including lung, bladder, colon and ovarian cancers. However no significant expression in normal tissue has been reported suggesting its potential use as a target for anti-STEAP1 immunotherapy. An anti-STEAP1 monoclonal antibody linked to an antimitotic agent is in Phase I clinical trials for prostate cancer patients. Microarray data from our lab suggested that STEAP1 is also highly expressed in human breast cancers. In this study we evaluate expression STEAP1 in primary tumors and the bone marrow from breast cancer patients as a therapeutic target for minimal residual disease. Experimental procedures: RNA was isolated from primary tumor and non-malignant breast tissue. RNA was also isolated from bone marrow of state II and III breast cancer patients, healthy volunteers and breast cancer cell lines. STEAP1 expression was analyzed by nanostring nCounter and real time qRT-PCR using human specific probes. STEAP1 immunohistochemical (IHC) staining of human tissue was performed using standard IHC Protocol. Results: STEAP1 mRNA was up-regulated in 77% of tumors (28/36) compared to the corresponding normal tissue. STEAP1 protein was expressed in 100% of tumors (8/8) and was absent in non-malignant breast tissue (7/7) by IHC staining. STEAP1 mRNA was not expressed normal BM, but was detected in 8% (6/74) of BM from patients with early stage breast cancer. Of patients with STEAP1 positive BM, 50% (3/6) had triple negative disease and 66% (4/6) developed recurrent disease (p = .028). Conclusion: STEAP1 is expressed in human breast tumors and the BM of breast cancer patients. Expression of STEAP1 in the BM is significantly associated with the development of metastatic disease. Our data indicate that STEAP1 could serve as a therapeutic target for the treatment of minimal residual disease in breast cancer. Citation Format: Chidananda Mudalagiriyappa, Sreeraj Pillai, Mark Watson, Rebecca Aft. Expression analysis of STEAP1 in breast cancer patients as therapeutic target. [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 5258. doi:10.1158/1538-7445.AM2015-5258

Abstract 364: The molecular profiles of disseminated tumor cells in a Patient Derived Xenograft model recapitulate those found in patient bone marrow

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objective Disseminated tumor cells (DTCs) found in the bone marrow (BM) of breast cancer patients portends a poor prognosis. BM DTCs are thought to be intermediaries in the metastatic process and may exhibit molecular features different from the primary tumor. Using Patient Derived Xenograft system, we report the presence of transcript associated with DTCs and their correlation to distant disease development. Experimental procedures After informed consent, human breast adenocarcinomas were prospectively collected from 5 patients with estrogen receptor negative/Her2 negative tumors and implanted into NOD/SCID mouse mammary fat pad as previous described. BM was collected from the femur and tibia from mice at varying passages of the tumors and analyzed for human-specific transcripts by qRT-PCR. Human gene expression array analysis (Affymetrix Human gene 1.0ST) was performed on the BMs from all WHIM17 mice, control non-tumor bearing mice, breast cancer patients and that of healthy volunteers. Results BM was screened from 18 tumor bearing mice for the presence of DTCs of which 10 mice developed metastatic tumors. All mice developed from one patient line (WHIM17) developed metastatic tumors. Further microanalysis of WHIM 17 BM showed 300 genes upregulated over 10 fold in their BM compared to non-tumor bearing mice. 7 of these genes were detected in the BM of breast cancer patients but completely absent in BM of healthy volunteers. They are TNFRSF17, CD226, HIST1H4E, MDM2, TFAM, DDT, and RPS20. The presence of these transcripts was confirmed by qRT-PCR and was detected in the BM of mice which developed metastatic tumors. Of the 32 genes which we found significantly upregulated in the BM of stage II/III breast cancer patients prior to treatment who subsequently developed metastatic tumors compared to similar patients who did not develop metastatic disease, DCSR3 and STAM2 were upregulated in all WHIM17 mice. Conclusion The presence of DTCs in the BM and its association with metastatic outcome was observed in the PDX model system. Our results provide an experimental support for the clinical association of DTCs in the BM of early stage breast cancer patients with recurrent disease development. We believe the PDX mice recreate a more clinically relevant model compared to other xenograft models. Moreover, using this system, we have identified new targetable genes associated with DTCS. Citation Format: Sreeraj G. Pillai, Shunqiang Li, Chidananda M. Siddappa, Mark A. Watson, Timothy P. Fleming, Matthew J. Ellis, Rebecca L. Aft. The molecular profiles of disseminated tumor cells in a Patient Derived Xenograft model recapitulate those found in patient bone marrow. [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 364. doi:10.1158/1538-7445.AM2015-364

Abstract 3082: Isolation and identification of disseminated tumor cells from bone marrow

Background: Disseminated tumor cells (DTCs), detected by immunohistological staining for cytokeratins, found in the bone marrow (BM) of breast cancer patients portend a poor prognosis. Study of these cells has been hindered by their rarity in BM specimens. We have tested a microfiltration technique for the enrichment of DTCs. Methods: BM aspirates from healthy volunteers were collected and mixed with defined numbers SKBR3 breast cancer cells at a concentration of 1,000 to 10,000 tumor cells per 1x10^6 nucleated BM cells., BM was then diluted 1:10 with PBS and 7 ml was placed in CellSave TM tubes to simulate a patient BM collection. Each specimen was processed within 24 hours of collection. Specimens were pre-filtered using a 70 µm mesh sieve (BD cat#352350) to remove large particles. The effluent was filtered through CellSieve TM microfilters, which have 7 µm diameter pores in a uniform array, of 160,000 pores in a 9 mm diameter area. Filters containing cells were post-fixed, cells permeabilized, and stained with DAPI and fluorescent antibodies specific to epithelial cytokeratins 8, 18, and 19 (FITC), EpCAM (PE), and CD45, a negative control (Cy5). Five representative 1% areas of each filter were imaged and used to estimate total cell and tumor cell counts, based on fluorescent DAPI and cytokeratin antibody staining. Results: From each BM specimen (7 ml) containing a total of 14x10^6 nucleated BM cells, post filtration samples contained only 17-21x10^3 residual, nucleated BM cells. For specimens spiked at 10 tumor cells per 1,000 BM cells, approximately 30x10^3 positively staining tumor cells were recovered (20% tumor cell recovery with approximately 170 fold enrichment). For specimens spiked at 1 tumor cell per 1,000 BM cells, approximately 7x10^3 total tumor cells were recovered (50% tumor cell recovery with approximately 400-fold enrichment). Captured cells co-stained with cytokeratins and EpCAM, but not CD45. Control BM specimens with no added tumor cells demonstrated only rare, staining cells (4 per sample), likely due to background or epithelial cell contamination. Conclusions: We have tested a microfiltration technique for enrichment of BM DTCs. Microfiltration of BM is rapid without filter clogging. Recovered DTCs can be immuno-stained, and are easily identified and distinguished from BM cells. The DTC capture efficiency varied with cancer cell concentration and ranged from 20-50% with up to 400 fold enrichment when samples contained lower numbers of tumor cells. The microfiltration enrichment assay continues to be optimized for isolation and molecular characterization of DTCs from patient BM samples, where the concentration of DTCs seldom exceeds 10 cells per 1x10^6 BM cells. Citation Format: Peixuan Zhu, Daniel Adams, Rebecca L. Aft, Sreeraj G. Pillai, Mark A. Watson, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang. Isolation and identification of disseminated tumor cells from bone marrow. [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 3082. doi:10.1158/1538-7445.AM2014-3082

Abstract P2-04-02: Identification of genes associated with breast cancer micrometastatic disease in bone marrow using a human-in-mouse xenograft system

Disseminated tumor cells (DTCs) found in the bone marrow (BM) of breast cancer patients portend a poor prognosis and are thought to be the intermediaries in the metastatic process. Study of these cells has been limited due to their scarcity. To develop a clinically relevant model to characterize hese cells, we have employed a human in mouse (HIM) xenograft model for propagating, isolating, and molecularly characterizing DTCs. Human breast adenocarcinomas were prospectively collected from 5 patients and implanted into humanized NOD/SCID mouse mammary fat pads. BM was collected from the long bones at varying passages of the tumors and analyzed for human-specific gene expression by qRT-PCR and gene expression microarray. Human-specific gene expression of SNAI1, GSC, FOXC2, KRT19, and STAM2 , presumably originating from disseminated tumor cells, was detectable in the BM of all mice that had developed metastatic disease to other solid organs, but was not detectable in xenotransplanted mice that did not develop metastatic disease. Comparative gene expression microarray analysis of the HIM primary tumor, the corresponding BM from mice with metastatic disease, and BM from control mice identified additional patterns of gene expression enriched in BM-associated DTCs which included several genes associated with epithelial-mesenchymal transition, aggressive clinical phenotype, and metastatic disease development in primary human tumors. We have found that BM DTCs can be detected using the HIM xenograft model and have identified unique patterns of gene expression associated with BM DTCs, which may provide further insight into the biology and therapeutic vulnerability of metastatic tumor cell populations in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-02.

Abstract 2234: Detection of minimal residual disease in the bone marrow of breast cancer patients using multiplex gene expression measurements with the Nanostring nCounter Assay

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Disseminated tumor cells (DTCs) are detected in up to 40% of breast cancer patients at the time of diagnosis and are an independent prognostic factor for recurrent disease. Presently DTCs are identified by immunohistochemical staining for cytokeratins and/or by molecular detection of single gene expression associated with DTCs. These diagnostic approaches are often subjective, laborious, and insensitive due to the molecular heterogeneity of the DTCs. In this study, we have determined whether a novel, multiplexed gene expression technology platform, Nanostring nCounterTM (NC), could be used for the multi-marker detection of DTCs. Methods: Total RNA was isolated from bone marrow (BM) of breast cancer patients, breast cancer cell lines, and healthy volunteers. RNA was analyzed directly with the NC assay or converted to cDNA and analyzed by qRT-PCR for DTC associated gene-expression. Results: Expression of 9 genes associated with breast cancer and DTC (TWIST1, PITX2, TACSTD1, SCGB2A2, EGFR, SNAI2, S100A3, KRT17 and KRT19) were examined by the NC assay and qRT-PCR. Using cell lines representing 3 main molecular breast tumor subtypes diluted into normal human BM at varying concentrations, we found that expression of KRT19, SNAI2, and EGFR could be simultaneously detected at a sensitivity of 1 cancer cell per 100,000 BM cells, using 0.5ug of input RNA. Expression measurements were quantitative, reproducible, and varied over a 20-fold linear dynamic range, depending on the cell line and its inherent expression of each transcript. Sensitivity was improved approximately 20-fold by Ficoll gradient enrichment of DTCs (a standard methodology for ICC-based detection) and increasing the amount of input RNA to 5 ug. This increased the sensitivity from 1 DTC per 1 million BM cells. The NC assay applied to BM collected prior to treatment from 5 patients with stage II/III breast cancer who had known DTCs detected by conventional ICC. 8 normal BM were used to establish baseline and threshold cutoff values. At least one of the nine genes in the multi-marker panel was detected in four of the five breast cancer patient BM samples. Interestingly, KRT19 itself was detected in only one specimen. The BM from two patients (ER-/Her2-) expressed genes known to be associated with this tumor type (TACSTD1, SNAI2, KRT17). Conclusion: Our data demonstrate the potentiality of the NC platform to detect multigene expression in rare cell populations like DTCs, with sensitivity equal to that of qRT-PCR. Using this approach, 1 DTC per 1 million BM cells can be detected, which is equal to the reported sensitivity of IHC detection (gold standard). Based on this data, we believe that the NC assay, with a more elaborated probe panel, will be a sensitive, specific, and diagnostically useful for the detection and molecular classification of DTCs in bone marrow from breast cancer patients. 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 2234. doi:10.1158/1538-7445.AM2011-2234

Abstract 1589: Development of a mouse model for bone marrow disseminated tumor cells with human tumor xenografts

Objective: Disseminated tumor cells (DTCs) found in the bone marrow (BM) of breast cancer patients portends a poor prognosis. BM DTCs are thought to be intermediaries in the metastatic process. Study of these cells has been limited due to their scarcity. To overcome this limitation, we have developed an animal model for DTCs employing the Human and Mouse Linked Evaluation of Tumor (HAMLET) system. Experimental procedures: After informed consent, human breast adenocarcinomas were prospectively collected from 5 patients with estrogen receptor negative/Her2 negative tumors and implanted into a humanized NOD/SCID mouse mammary fat pad as previous described. BM was collected from the femur and tibia from mice at varying passages of the tumors and analyzed for human-specific GADPH (hGAPDH) expression by qRT-PCR. The presence of other human gene transcripts, previously detected in the BM of breast cancer patients and believed to be associated with the presence of DTCs, was also determined by qRT-PCR. Human gene expression array analysis (Affymetrix Human gene 1.0ST) was performed on the primary xenograft tumor, a spleen metastatic nodule and BM from all WHIM17 mice. Results: BM was screened from 16 tumor bearing mice for the presence of DTCs. 10 animals, with tumors derived from 2 patients, developed metastatic disease and 9 had detectable levels of hGAPDH. The CT ratio human to mouse GAPDH varied from 1.06 to 2.23. In particular, high levels of hGAPDH were found in one HAMLET mouse line, WHIM 17, in which all animals developed metastases (CT ratio human to mouse GAPDH=1.06 to 1.31). One mouse with detectable hGAPDH died of other causes and could not be assed for metastatic disease development. There was no detectable hGAPDH in five mice, derived from 3 patient tumors, which did not develop metastatic tumors and in control mice with humanized mammary fat pads. Human-specific transcripts for SNAIL1, GSC, FOXC2 and KRT19 were detectable in the BM of the WHIM 17 mice whereas control, non-tumor bearing humanized mice demonstrated no expression of any of these markers. Microarray analysis confirmed the presence of human cells in mouse BM, allowing the identification of genes specifically associated with BM DTC cell populations. Conclusion: We have utilized a mouse xenograft model for the study of DTCs using primary human breast adenocarcinomas transplanted into NOD/SCID mice. The presence of human cells in mice BM appears to correlate with metastatic tumor development. The gene expression patterns of these cells indicate that they maintain molecular profiles similar to DTCs isolated directly from the BM of breast cancer patients. We believe that this model will allow for a better understanding of the metastatic process in breast cancer patients and provide an in vivo model for monitoring and assessing the efficacy of new therapeutic agents in eradicating micrometastatic disease and ultimately, improving survival in breast cancer patients. 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 1589. doi:10.1158/1538-7445.AM2011-1589

P4-06-03: Multiplex Gene Expression of Disseminated Tumor Cells in the Bone Marrow of Breast Cancer Patients Identifies Novel Therapeutic Targets.

Background: Disseminated tumor cells (DTCs) are detected in the bone marrow (BM) of up to 40% of breast cancer patients at the time of diagnosis and are an independent prognostic factor for recurrent disease. Present techniques for detection of DTC are often laborious, and insensitive due to the molecular heterogeneity of the DTCs. We have previously optimized and validated a novel, multiplexed gene expression technology platform, Nanostring nCounter™ (NC) which counts single molecules of RNA, for the multi-marker detection of DTCs in BM at a sensitivity of 1 cancer cell per 1 million nucleated BM cells. We now validate a 36 gene panel for the detection and molecular characterization of DTCs in BM. Methods : Hybridization probes for 36 genes whose expression are associated with breast cancer, metastasis, and/or the cancer stem cell phenotype, and which exhibit no or low expression levels in normal bone marrow by qRT-PCR were developed for the NC assay. Total RNA was isolated from whole BM collected from the right and left iliac crest from breast cancer patients and healthy volunteers. 5 ug of RNA was analyzed, in duplicate with the NC assay. BM was scored positive for expression of an individual gene if expression in duplicate samples was 2 standard deviations above mean expression in a set of 11 independent normal BM samples. Results: Bilateral BM samples were analyzed prior to any therapy from 20 patients: 8 developed metastatic disease within 2–48 months (mean of 23 months) after diagnosis, and 12 had no evidence of metastatic disease with 3–5 years follow-up. Overall, expression of at least one gene in the 36-gene multi-marker panel was detected in 17 patients (85%). There was excellent correlation between individual gene expression in both the right and left iliac crest samples from the same patient. Six of the 8 patients (75%) who developed metastatic disease had detectable expression of 1–3 genes. Two genes were commonly associated with metastatic disease development. 50% (3 of 6) of the patients who had detectable expression of EBB2 in their BM developed metastatic disease, although this did not correlate with expression in the corresponding primary tumor from the same patient. 80% (4 of5) of the patients who expressed the hedgehog pathway gene, Ptch1, in their BM developed metastatic disease. Conclusions: Our data demonstrate the feasibility of using a 36-plex NC assay to detect gene expression associated with BM DTCs in breast cancer patients. We found expression of 2 targetable genes associated with the development of metastatic disease, ERBB2 and Ptch1. ERBB2 expression in BM did not correlate with expression in the primary tumor. The molecular diversity of gene expression observed underscores the need for a multiplexed gene expression panel. Ongoing studies are evaluating the clinical utility of this assay to detect DTCs relative to existing techniques, for predicting relapse-free survival, molecular classification, and selecting appropriate targeted therapeutics based on BM DTC profiles in breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-03.