Bone Marrow Of Breast Cancer Patients Research Articles

CXCR4 and JUNB double-positive disseminated tumor cells are detected frequently in breast cancer patients at primary diagnosis.

Background:The chemokine receptor CXCR4 and the transcription factor JUNB, expressed on a variety of tumor cells, seem to play an important role in the metastatic process. Since disseminated tumor cells (DTCs) in the bone marrow (BM) have been associated with worse outcomes, we evaluated the expression of CXCR4 and JUNB in DTCs of primary, nonmetastatic breast cancer (BC) patients before the onset of any systemic treatment.Methods:Bilateral BM (10 ml) aspirations of 39 hormone receptor (HR)-positive, HER2-negative BC patients were assessed for the presence of DTCs using the following combination of antibodies: pan-cytokeratin (A45-B/B3)/CXCR4/JUNB. An expression pattern of the examined proteins was created using confocal laser scanning microscopy, Image J software and BC cell lines.Results:CXCR4 was overexpressed in cancer cells and DTCs, with the following hierarchy of expression: SKBR3 > MCF7 > DTCs > MDA-MB231. Accordingly, the expression pattern of JUNB was: DTCs > MDA-MB231 > SKBR3 > MCF7. The mean intensity of CXCR4 (6411 ± 334) and JUNB (27725.64 ± 470) in DTCs was statistically higher compared with BM hematopoietic cells (2009 ± 456, p = 0.001; and 11112.89 ± 545, p = 0.001, respectively). The (CXCR4+JUNB+CK+) phenotype was the most frequently detected [90% (35/39)], followed by the (CXCR4–JUNB+CK+) phenotype [36% (14/39)]. However, (CXCR4+JUNB–CK+) tumor cells were found in only 5% (3/39) of patients. Those patients harboring DTCs with the (CXCR4+JUNB+CK+) phenotype revealed lower overall survival (Cox regression: p = 0.023).Conclusions:(CXCR4+JUNB+CK+)-expressing DTCs, detected frequently in the BM of BC patients, seem to identify a subgroup of patients at higher risk for relapse that may be considered for close follow up.

Abstract LB-312: Interleukin 6 transsignaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency

Abstract While thousands of breast cancer cells disseminate and home to bone marrow (BM) until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. Signals and mechanisms determining failure or success of disseminated cancer cells (DCCs) are largely unknown and there is no in vivo model available to study the spontaneous progression and genomic evolution from early bone marrow infiltration to manifestation of bone metastasis, as spontaneous or transgenic mouse models do not generate bone metastases. We therefore profiled DCCs from BM of breast cancer patients long before manifestation of metastasis by RNAseq to identify signals supporting survival or outgrowth of DCCs and identified IL6/PTEN/PI3K signaling as candidate pathway for DCC activation. Since early DCCs often display close-to-normal genomes we used mammary epithelial cells ex vivo isolated from reduction mammoplasties and immortalized pre-malignant breast cancer cell lines as model for functional testing in vitro. Using specific activators and inhibitors of IL6 signaling revealed that IL6 trans, but not classical signaling, regulates stemness of mammary epithelial cells. Moreover, knock-down of PTEN revealed that PI3K/PTEN pathway activation renders cells independent of IL6 trans-signaling. Interestingly, gp130 expression, a pre-requisite for IL6 trans-signaling was found to be down-regulated by bone marrow stromal and endosteal, but not vascular niche cells, and as a consequence the number of cells with stem-like ability was significantly reduced. Consistent with a bottleneck function of microenvironmental DCC control, we found PIK3CA mutations highly associated with late-stage metastatic DCCs and CTCs while generally absent in early DCCs. Our data suggest that the initial steps of metastasis formation depend on microenvironmental signals and are not cancer cell-autonomous. Citation Format: Melanie Werner-Klein, Ana Grujovic, Milan Obradovic, Martin Hoffmann, Xin Lu, Stefan Kirsch, Steffi Treitschke, Cäcilia Köstler, Kathrin Weidele, Christoph Irlbeck, Catherine Botteron, Christian Werno, Bernhard Polzer, Miodrag Guzvic, Stefan Buchholz, Petra Rümmele, Norbert Heine, Stefan Rose-John, Christoph A. Klein. Interleukin 6 transsignaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-312.

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.

Tumor specific regulatory T cells in the bone marrow of breast cancer patients selectively upregulate the emigration receptor S1P1

Regulatory T cells (Treg) hamper anti-tumor T-cell responses resulting in reduced survival and failure of cancer immunotherapy. Among lymphoid organs, the bone marrow (BM) is a major site of Treg residence and recirculation. However, the process governing the emigration of Treg from BM into the circulation remains elusive. We here show that breast cancer patients harbour reduced Treg frequencies in the BM as compared to healthy individuals or the blood. This was particularly the case for tumor antigen-specific Treg which were quantified by MHCII tumor peptide loaded tetramers. We further demonstrate that decreased Treg distribution in the BM correlated with increased Treg redistribution to tumor tissue, suggesting that TCR triggering induces a translocation of Treg from the BM into tumor tissue. Sphingosine-1-phosphate receptor 1 (S1P1)—which is known to mediate exit of immune cells from lymphoid organs was selectively expressed by tumor antigen-specific BM Treg. S1P1 expression could be induced in Treg by BM-resident antigen-presenting cells (BMAPCs) in conjunction with TCR stimulation, but not by TCR stimulation or BMAPCs alone and triggered the migration of Treg but not conventional T cells (Tcon) to its ligand Sphingosine-1-phosphate (S1P). Interestingly, we detected marked S1P gradients between PB and BM in breast cancer patients but not in healthy individuals. Taken together, our data suggest a role for S1P1 in mediating the selective mobilization of tumor specific Treg from the BM of breast cancer patients and their translocation into tumor tissue.

Paired-like Homeodomain Transcription factor 2 expression by breast cancer bone marrow disseminated tumor cells is associated with early recurrent disease development.

The presence of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients is prognostic for early relapse. In the present study, we analyzed the gene expression profiles from BM cells of breast cancer patients to identify molecular signatures associated with DTCs and their relevance to metastatic outcome. We analyzed BM from 30 patients with stage II/III breast cancer by gene expression profiling and correlated expression with metastatic disease development. A candidate gene, PITX2, was analyzed for expression and phenotype in breast cancer cell lines. PITX2 was knocked down in the MDAMB231 cell lines for gene expression analysis and cell invasiveness. Expression of various signaling pathway molecules was confirmed by RT–PCR. We found that the expression of Paired-like Homeobox Transcription factor-2 (PITX2) is absent in the BM of normal healthy volunteers and, when detected in the BM of breast cancer patients, is significantly correlated with early metastatic disease development (p = 0.0062). Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells. Three genes—NKD1, LEF1, and DKK4—were significantly downregulated in response to PITX2 suppression. Expression of PITX2 in BM of early-stage breast cancer patients is associated with risk for early disease recurrence. Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-015-3576-z) contains supplementary material, which is available to authorized users.

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

Abstract 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 4076: Characterizing tumor-specific memory stem like T cells in blood and bone marrow of breast cancer patients

Abstract The identification of memory T cells with stem cell-like properties has been reported recently in both humans and mice. Such stem cell-like memory T cells (Tscm) upon adoptive transfer into a humanized mouse model had superior self-renewal, proliferation and mediated potent anti tumor immunity. However, very little is known about their specificities, physiological function and contribution to anti-tumor immune responses in cancer patients. We investigated the presence of Tscm cells in the peripheral blood (PB) and bone marrow (BM) of breast cancer patients by multicolor flow cytometry analyses with a panel of human T cell markers to distinguish naïve (Tn), central memory (Tcm), effector memory (Tem) and Tscm (CCR7+, CD45RO-, CD127+, CD45RA+, CD95+, CXCR3+, IL2Rβ+) population. Tscm cells were detected in both CD4+ and CD8+ T cells in the PB and BM of breast cancer patients. Interestingly, we observed a significant increase (3-fold) in the number of Tscm cells among the CD4+ and CD8+ T cells in breast cancer patients as compared to healthy donors. This was significantly higher among the CD8+ T cells as compared to CD4+ T cells. We further evaluated the expression of exhaustion markers (PD-1 & LAG3) and found that majority of Tscm cells (>95%) did not express PD-1 & LAG3 and comprised the least exhausted T cell compartment as compared to Tn, Tem and Tcm. To investigate the contribution of different subsets of memory T cells to tumor-specific T cells in breast cancer patients, we used a panel of breast cancer associated MHC class-I pentamers against human epidermal growth factor receptor 2 (HER2348-356 , HER2435-443), Mucin-1 (MUC112-20) and Carcinoembryonic antigen (CEA571-579). Cumulative analyses of tumor-specific pentamer+ CD8+ T cells in PB and BM showed that 20-25% of total tumor-specific T cells were of Tscm phenotype. We observed higher percentages of tumor-specific pentamer+CD8+ T cells among the total Tscm cells in BM as compared to PB, however this difference was not significant. We performed ex vivo re-stimulation assays using autologous dendritic cells loaded with several breast cancer-specific peptides with PB and BM of breast cancer patients to further assess the frequency and function of tumor-specific Tscm cells. Cumulative analyses showed that 20-23% of tumor-specific IFN-γ+ CD8+ and 26-32% IL-2+ CD4+ had a Tscm phenotype. In summary, we report the presence of Tscm cells in the PB and BM of breast cancer patients and a significant increase in their number as compared to healthy donors. Importantly, we could show that Tscm contribute to the population of functional and tumor reactive CD8+ and CD4+ T cells in PB and BM of breast cancer patients. Our further aim is to investigate the therapeutic potential of Tscm cells in adoptive T cell experiments with humanized mouse model of breast cancer. Citation Format: Mudita Pincha, Paranchai Boonsawat, Christoph Domschke, Philipp Beckhove. Characterizing tumor-specific memory stem like T cells in blood and bone marrow of breast cancer patients. [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 4076. doi:10.1158/1538-7445.AM2014-4076

MUC2 mRNA detection in peripheral blood and bone marrow of breast cancer patients reveals micrometastasis

Tumor dissemination to distant organ is the main cause of death. Therefore there is urgent need to set up sensitive methods for early detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in peripheral blood (PB) and bone marrow (BM) specimens of breast cancer patients. We aim to detect MUC2 mRNA positive cells in PB and BM of breast cancer patients; to relate this to patient relapse. In this study to detect MUC2 mRNA positive cells (tumor marker), PB and BM samples were collected from 50 breast cancer patients after operation and before adjuvant therapy with 20 PB from healthy individuals as negative controls. Chi-square test was used to analyze data. MUC2 mRNA by using Real-time PCR was detected in 9 (18%) of PB and in 10 (20%) of BM samples and none of the healthy individuals. The relapse rate among MUC2-positive patients was significance in BM (P

Detection of disseminated tumor cells in the bone marrow of breast cancer patients using multiplex gene expression measurements identifies new therapeutic targets in patients at high risk for the development of metastatic disease

Disseminated tumor cells (DTCs) detected in the bone marrow (BM) of breast cancer patients identify women at high risk of recurrence. DTCs are traditionally detected by immunocytochemical staining for cytokeratins or single gene expression measurements, which limit both specificity and sensitivity. We evaluated the Nanostring nCounter™ platform for multi-marker, gene expression-based detection and classification of DTCs in the BM of breast cancer patients. Candidate genes exhibiting tumor cell-specific expression were identified from microarray datasets and validated by qRT-PCR analysis in non-malignant human BM and identical samples spiked with predefined numbers of molecularly diverse breast tumor cell lines. Thirty-eight validated transcripts were designed for the nCounter™ platform and a subset of these transcripts was technically validated against qRT-PCR measurements using identical spiked BM controls. Bilateral iliac crest BM aspirates were collected and analyzed from twenty breast cancer patients, prior to neoadjuvant therapy, using the full 38-gene nCounter™ code set. Tumor cell-specific gene expression by nCounter™ was detected with a sensitivity of one cancer cell per 1 × 10(6) nucleated BM cells after optimization. Measurements were quantitative, log linear over a 20-fold range, and correlated with qRT-PCR measurements. Using the nCounter™ 38-gene panel, 6 of 8 patients (75 %) who developed metastatic disease had detectable expression of at least one transcript. Notably, three of these patients had detectable expression of ERBB2 in their BM, despite the fact that their corresponding primary tumors were HER2/ERBB2 negative and therefore did not receive trastuzumab therapy. Four of these patients also expressed the PTCH1 receptor, a newly recognized therapeutic target based on hedgehog signaling pathway inhibition. The presumptive detection and classification of DTCs in the BM of breast cancer patients, based on sensitive and quantitative multi-marker detection of gene expression using the nCounter™ platform, provide an opportunity to both predict early distant recurrence and, more importantly, identify opportunities for preventing the spread of disease based on the expression of unique, therapeutically actionable gene targets. This study demonstrates the application of a new technology for multiplexed gene expression-based detection of DTCs in the BM of breast cancer patients and identifies at least two therapeutically targetable genes that are frequently expressed in the BM of patients who develop metastatic disease.

Epithelial cells expressing cytokeratins-19 and bone marrow micrometastases in patients with breast cancer at the time of primary surgery: clinical outcome during long-term follow-up

To evaluate the detection of epithelial cells in bone marrow of breast cancer patients as an indicator of metastatic disease. Between 2001 and 2005, bone marrow biopsies were taken from 79 breast cancer patients during primary surgery. Specimens were stained immunocytochemically for epithelial cells expressing cytokeratins or epithelial membrane antigen. The long-term outcomes of these patients were analyzed. In 51 CK-positive results of 79 patients, epithelial cells were found in the bone marrow (BM) biopsies. These patients were designated CK(+). The rate of tumor recurrence or cancer-related death was significantly higher in CK(+) patients than in CK-negative patients. Multivariate analysis using the Cox regression model revealed BM status as a prognostic parameter independent of axillary lymph node status. Disseminated epithelial cells in BM are associated with poor clinical outcome in breast cancer patients. However, the presence of these cells is not a sufficient parameter, suggesting that epithelial cells in the BM of breast cancer patients at the time of surgery have limited metastatic potential. The role of these cells needs to be further evaluated.

Prognostic relevance of induced and spontaneous apoptosis of disseminated tumor cells in primary breast cancer patients.

e21003 Background: An imbalance between cell proliferation and programmed cell death leads to tumor growth. Although most cytostatic agents used in systemic therapy of breast cancer induce an apoptosis in tumor cells leading to their destruction, a high ratio of apoptotic cells in primary breast cancer is correlated with poor prognosis. The purpose of this study was to investigate the incidence and prognostic significance of apoptotic disseminated tumor cells (DTC) in bone marrow (BM) of primary breast cancer (PBC) patients. Methods: PBC patients who underwent primary surgery (PS) or were treated with neoadjuvant chemotherapy (NACT) between 01/2003 and 12/2007 were included into this prospective study. BM aspirates were analyzed by immunocytochemistry (M30 antibody) using ACIS system (Chromavision) according to the ISHAGE evaluation criteria. Patients without DTC in the BM were excluded from the study. Survival data were evaluated after a median follow up of 44 months. Results: BM aspirates of 383 PBC patients were analyzed. DTC were M30 positive in 82 of 298 (27%) patients who underwent PS and 41 of 85 (48%) patients treated with NACT. After NACT, the incidence of apoptotic cells in patients with complete/partial remission as compared to patients with stable/progressive disease was 63%/53% vs. 31%/0% (p=0.034). In the NACT group, M30-positive patients suffered less likely of a relapse than those without apoptotic DTC (7% vs. 23% of the events, p = 0.049). Inversely, overall survival in the PS group was significantly shorter in M30-positive as compared to M30-negative patients (75 months, 95% CI: 68-81 months vs. 84 months, 95% CI: 81-86 months; p = 0.008). Conclusions: Apoptotic DTC can be detected in breast cancer patients before and after systemic treatment. The presence of apoptotic DTC in BM of breast cancer patients can reflect the effects of neoadjuvant treatment. Spontaneous and chemotherapy-induced apoptosis may have different prognostic implications.

Expression of human mammaglobin as a marker of bone marrow micrometastasis in breast cancer

The aim of the present study was to detect the expression of human mammaglobin (hMAM) mRNA in the bone marrow (BM) of patients with breast cancer and determine the relationship between micrometastasis and clinicopathological parameters as well as selected molecular markers and breast cancer prognosis. The expression of hMAM mRNA in the BM of patients with breast cancer was determined by RT-PCR. The expression of ER, PR and Cath-D in cancer tissues was detected by immunohistochemistry. A positive expression rate for hMAM of 38.2% in 102 patients with stage I-III breast cancer was found. The expression of hMAM was higher in patients with T(2-3) (>2 cm) tumors than in those with T(1) tumors (≤2 cm) (χ(2)=19.20, P=0.001) and in patients with stage II or III tumors than in patients with stage I tumors (χ(2)=15.101, P=0.001). The expression of hMAM in the BM of breast cancer patients categorized as grade 1 was lower than that in those of grade 2 or 3 (χ(2)=8.522, P=0.014), and hMAM expression was related to the pathological type of tumor (χ(2)=6.892, P=0.032) and the degree of axillary lymph node metastasis (χ(2)=14.050, P=0.001). The expression of hMAM in BM was much higher in patients with ER(-) or ER(+) tumors than in those with ER(++ or +++) (χ(2)=11.800, P=0.003), and those with PR (χ(2)=8.759, P=0.013). hMAM expression in BM was also significantly positively correlated with Cath-D expression (χ(2)=6.623, P=0.036). However, no correlation was found between hMAM expression and patient age (χ(2)=1.056, P=0.304). There was a strong correlation between patients with positive expression of hMAM in the BM and the presence of distant metastases (P=0.009). In conclusion, micrometastasis in the BM correlates with certain clinical pathological parameters and several tumor markers. Patients with positive expression of hMAM in the BM have a greater chance of distant metastasis and poor prognosis. The detection of micrometastasis may be one of the most advantageous markers for predicting the prognosis of breast cancer.

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

Abstract 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

Diagnostic accuracy of small breast epithelial mucin mRNA as a marker for bone marrow micrometastasis in breast cancer: a pilot study

Detection of isolated tumour cells (ITC) in the blood or minimal deposits in distant organs such as bone marrow (BM) could be important to identify breast cancer patients at high risk of relapse or disease progression. PCR amplification of tissue or tumour selective mRNA is the most powerful analytical tool for detection of this micrometastasis. We have evaluated for the first time, the diagnostic accuracy of small breast epithelial mucin (SBEM) as a potential marker for BM micrometastasis in breast cancer. A nested RT-PCR assay for detection of SBEM mRNA was compared with immunocytochemistry (ICC) with anticytokeratin AE1/AE3 antibody in paired samples obtained from the BM of breast cancer patients. Associations of SBEM mRNA detection in BM and clinical and pathological parameters were evaluated. SBEM mRNA status and time to breast cancer progression were analysed using Kaplan-Meyer curves. Fifty stages I-IV breast cancer female patients were prospectively included in our study. SBEM specific transcript was found in BM in 26% of the patients. Detection rate was similar to the percentage of patients with ITCs detected using ICC (24%). SBEM mRNA in BM aspirates were significantly associated with presence of clinically active disease, including locally advanced and metastatic patients (47%, P = 0.021) and tumours with positive hormonal receptors (36.7%, P = 0.035). In addition association with Her2/neu over-expression (44.4%, P = 0.051) and low proliferating tumours (36%, P = 0.067) were close to significant levels. When we analysed time to breast cancer progression adjusting for grade or hormone receptor status, presence of SBEM mRNA in BM defines distinct prognostic groups. SBEM might represent a suitable marker for molecular detection of ITCs in BM in breast cancer patients. Analysis of prognostic value for SBEM mRNA-based assay should take into account the heterogeneity and different molecular subtypes of breast cancer.

Detection and characterization of disseminated tumor cells present in bone marrow of cancer patients

Early tumor cell dissemination occurs even in patients with small solid tumors, and bone marrow (BM) is a common homing organ for blood-borne disseminated tumor cells (DTCs) derived from primary tumors. Immunocytochemical or molecular assays allow the detection of a single DTC in BM at a frequency of one tumor cell in one million surrounding hematopoietic cells; e.g., tumor cells are frequently detected in the BM of breast cancer patients without clinical or even histopathologic signs of metastasis. Evidence has emerged that the detection of DTCs and circulating tumor cells (CTCs) in blood may provide important prognostic information and, in addition, might help to monitor efficacy of therapy.

Estrogen receptor (ER) status of disseminated tumor cells in the bone marrow of breast cancer patients

21013 Background: The presence of disseminated tumor cells (DTC) in bone marrow (BM) of primary breast cancer patients is associated with poor prognosis. These patients may benefit from adjuvant endocrine therapy since cytotoxic agents are not able to completely eliminate DTCs as previously shown. Only patients with hormone receptor positive breast cancer are eligible for hormonal treatment. The ERa status is routinely defined in primary tumor tissue. However, the ERa status of DTC may differ compared to the primary tumor. Therefore, the aims of this study were (1) to determine the ERa status of DTC in BM of breast cancer patients, (2) and to compare the ERa status of DTC and corresponding primary tumors. Methods: BM aspirates from 251 primary breast cancer patients were included into the study. A double immunofluorescence staining procedure was established for the identification of cytokeratin-positive (CK)/ERa positive cells. ERa status of the primary tumor was immunohistochemically assessed using the same antibody against ERa. Results: In 105 of 251 (42%) breast cancer patients CK-positive cells could be detected in BM. The number of detected cells ranged between 1 and 13 / cells per 2*106 mononuclear cells. Disseminated tumor cells demonstrated ERa positivity in 13 (12%) of these 105 patients. The ERa expression on DTC was heterogeneous in 10 of 13 (79%) patients. Concordance rate of ERa status between primary tumor and DTC was 27%. Only 11 of 83 patients with ER a positive tumors had also ERa positives DTC. Conclusions: (1)The hormone receptor status between primary tumor and corresponding DTC is disconcordant. (2)This discrepancy may explain the rate of non-responders to adjuvant endocrine therapy despite ER-positive primary tumors. (3)These patients may benefit from adjuvant therapy regimens based on antibody strategies or bisphosphonates. No significant financial relationships to disclose.

Bone marrow cytokeratin 19 mRNA level is an independent predictor of relapse-free survival in operable breast cancer patients

To study the prognostic significance of elevated cytokeratin 19 (CK19) mRNA levels in the bone marrow (BM) of operable breast cancer patients. From 1998 to 2000, BM was collected from 195 consecutive breast cancer patients immediately prior to surgery and from 34 healthy volunteers. The patients received surgical and adjuvant treatment according to national guidelines at the time. We analyzed the level of CK19 mRNA in the BM samples from patients and normal controls using a real-time RT-PCR assay. The associations with known prognostic factors and the impact of pathological CK19 mRNA levels on patients' prognosis were investigated. Using the 99 percentile of the normal control group as a cut-off, 24 (12%) of the 195 patients and 1 (3%) of the 34 volunteers were diagnosed as CK19 mRNA positive. There was no correlation between CK19 BM status and the clinicopathological factors tested. During a median follow-up of 72 months, 7 (29%) of the 24 CK19 mRNA BM positive patients experienced systemic relapse compared to 20 (12%) of the 171 in the CK19 mRNA negative group. The patients with CK19 mRNA-positive BM had significantly shorter systemic recurrence-free survival (P=0.01) and overall recurrence-free survival (P=0.005). Multivariate Cox regression showed CK19 mRNA BM status to be an independent predictor of relapse. Detection of CK19 mRNA in the BM of breast cancer patients by real-time RT-PCR is an independent predictor of relapse-free survival in operable breast cancer patients.

Persistence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients predicts increased risk for relapse—Results of pooled European data

10083 Background: The prognostic significance of DTC in the BM of breast cancer patients at the time of primary diagnosis has recently been confirmed by a large pooled analysis. If the persistence of DTC after adjuvant therapy confers a similar risk for relapse, there might be an indication for secondary adjuvant treatment. Methods: We analyzed BM aspirates of 697 patients from academic breast cancer units in Oslo (n=356), Munich (n=228) and Tuebingen (n=113) during recurrence-free follow-up at a median interval of 32.4 months (standard deviation [std] 19.4 mon) after primary diagnosis of breast cancer pT1–4, pN0–3 pM0. Carcinoma cells were detected using a standardized immunoassay with the monoclonal antibodies A45-B/B3 (Munich, Tuebingen), or AE1 and AE3 (Oslo), directed against cytokeratin (CK). Patients were followed for a median of 54.2 months (std 24.5 mon) after primary diagnosis. Results: Persistent DTC in the BM were detected in 15.6% of the patients (n=109). The Kaplan-Meier estimate for mean distant relapse-free survival estimate was 155.6 mon (142.4 - 168.9 95%CI) in patients with negative and 102.3 mon (93.6 - 111.0, 95% CI, p< .0001, log rank test) in patients with positive BM status. Patients without evidence of persistent DTC had a significantly longer overall survival (164.4 [155.6 - 173.3]), than patients with positive BM status (101.7 mon [89.4 - 113.9], p< .0001). In multivariate Cox regression analysis, allowing for bone marrow status, tumor size, nodal status, histopathological grading and hormone receptor status, DTC was of higher independent prognostic significance for subsequent reduced breast cancer specific survival (RR 5.9, 2.8 - 12.8, 95% CI, p< .0001), than nodal status at time of primary diagnosis (RR 1.2, 1.0 - 1.3, 95% CI, p=.014). Conclusion: Evidence of persistent DTC in breast cancer patients indicates an increased risk for subsequent relapse, and may serve for monitoring in future clinical trials. Such trials might investigate the benefit of individualized secondary adjuvant treatment or extended adjuvant therapy of patients with DTC. No significant financial relationships to disclose.

Comparison of HER2 status between primary tumor and disseminated tumor cells in␣primary breast cancer patients

The persistence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients is associated with poor prognosis. Preliminary studies indicated that these patients might benefit from secondary adjuvant targeted therapy. HER2 protein is suggested as one of the most promising targets. The aims of this study were (1) to determine the HER2 status of DTC in BM of breast cancer patients and (2) to compare the HER2 status of DTC and corresponding primary tumors. BM aspirates from 137 primary breast cancer patients were included into the study. A double staining procedure was used for the identification of cytokeratin-positive (CK)/HER2 positive cells. HER2 status of the primary tumor was immunohistochemically assessed by the HERCEP-test. In 46 of 137 (34%) breast cancer patients CK-positive cells were detectable in BM. DTC with HER2 positivity were found in 20 (43%) of these patients. The HER2 expression on DTC was heterogeneous in 7 of 17 (41%) patients. Concordance rate of HER2 status between primary tumor and DTC was 62%. In 12 of 20 patients with HER2 negative tumors HER2 positive DTC were detected. HER2 positive DTC can be detected in patients with HER2 negative primary tumors. Therefore, the antigenic profile of DTC may be considered for treatment decision since these patients might actually benefit from trastuzumab. However, the HER2 overexpression on DTC is heterogeneous in individual patients which may reduce the efficacy of an immunotherapy based strategy directed against HER2-antigen only.

HER2 expression in disseminated tumor cells (DTC) and corresponding primary tumors of breast cancer patients

9580 Background: The presence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients is associated with poor prognosis. Several studies have indicated that these patients may benefit from secondary adjuvant immunotherapy. HER2 protein is suggested as one of the promising targets. The phenotype of the primary tumor is the current gold standard to make antibody based treatment decisions. However, the antigenic profile of primary tumors and DTC may be different. The aims of this study were (1) to determine the HER2 status of DTC in BM of breast cancer patients (2) and to compare the HER2 status of DTC and corresponding primary tumors. Method: Cytospins from bone marrow aspirates of 124 primary breast cancer patients (stage I-III) were analyzed for this study. A double labeling procedure was used for the identification of cytokeratin-positive (CK+)/HER2 positive cells. Slides were incubated with the pan-anti-cytokeratin antibody C11 conjugated to FITC and the polyclonal rabbit antibody CB11 against the HER2 protein. CB11 was detected by a secondary antibody labeled with Texas Red. HER2 status of the primary tumor was immunohistochemically assessed by the HERCEP-test (Dako Co., CA). Results: Disseminated tumor cells (DTC) were detected in the bone marrow of 47 patients. The number of DTC ranged from 1 to 70 per slide. 24 of these patients had HER2 positive DTC. HER2 positivity (score: 2+/3+) could be demonstrated in 12 of 46 primary tumors. Concordance rate between primary tumor and DTC was 63%. In three patients with HER2 positive tumors HER2 negative DTC were detected. In contrast, 14 patients with HER2 negative tumors (score: 0/1+) had HER2 positive DTC. The HER2 expression in DTC was heterogeneous in 8 of 20 patients with more than one CK-positive cell. Conclusions: (1) HER2 positive DTC can be detected in patients with HER2 negative primary tumors. Therefore, the antigenic profile of DTC should be included in the treatment decision for antibody based strategies. (2) The DTC are heterogeneous with regard to HER2 positivity. The effect of heterogeneity on the patient’s response is yet to be determined. No significant financial relationships to disclose.