Breast Cancer Bone Marrow Research Articles

Microfluidic Isolation of Disseminated Tumor Cells from the Bone Marrow of Breast Cancer Patients.

Disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients are putative precursors of metastatic disease, and their presence is associated with an adverse clinical outcome. To achieve the personalization of therapy on a clinical routine level, the characterization of DTCs and in vitro drug testing on DTCs are of great interest. Therefore, biobanking methods, as well as novel approaches to DTC isolation, need to be developed. In this study, we established a protocol for the biobanking of BM samples and evaluated a microfluidic-based separation system (Parsortix®) for the enrichment of cryopreserved DTCs. We were able to successfully isolate viable DTCs after the prior cryopreservation of BM samples. We calculated a significant increase of up to 90-fold in harvested DTCs with the proposed method compared to the current standard techniques, opening up new analysis possibilities for DTCs. Our advanced method further presents options for 3D DTC cultures, enabling the individualized testing of targeted therapies for BC patients. In conclusion, we present a novel approach for DTC enrichment, with possibilities for future clinical implications.

Low RAI2 expression is a marker of poor prognosis in breast cancer.

Retinoic acid-induced 2 (RAI2) has been shown to be a putative suppressor of the early hematogenous dissemination of tumor cells to the bone marrow in breast cancer. Here, we investigated the associations of RAI2 mRNA and protein expression with clinicopathological factors and prognosis in breast cancer patients with long-term follow-up. Invasive breast cancer tissues (n = 604) were analyzed for RAI2 mRNA expression. We examined the associations of clinicopathological factors with the expression levels of RAI2 mRNA in these samples. We also analyzed RAI2 protein expression by immunohistochemistry in invasive breast cancer tissues (n = 422). We identified significant positive associations between low expression of RAI2 mRNA and shorter disease-free survival (DFS), breast-cancer-specific survival (BCSS), and overall survival (OS) in breast cancer patients. We also identified significant positive associations between negative for RAI2 protein expression and shorter DFS, BCSS, and OS in breast cancer patients. Low RAI2 mRNA and negative for RAI2 protein expression were positively associated with larger tumor size, higher tumor grade, and ERα-negativity. Multivariate analyses indicated that not only RAI2 mRNA but also RAI2 protein expression were independent risk factors for both DFS and BCSS in breast cancer patients. The median follow-up periods were 10.3 and 9.3years for the RAI2 mRNA and protein expression analyses, respectively. Our findings suggest that RAI2 has a role in the metastasis of breast cancer, and that RAI2 expression could be a promising candidate biomarker of prognosis in breast cancer patients.

Influence of denosumab on disseminated tumor cells (DTC) in the bone marrow of breast cancer (BC) patients with neoadjuvant treatment: A GeparX translational substudy.

580 Background: DTCs in the bone marrow are observed in up to 30% at primary diagnosis of BC and their presence is an independent prognostic factor for reduced survival. It was shown that antiresorptive therapy eradicates DTCs and improves prognosis in DTC-positive BC patients (pts). In the GeparX phase III prospective randomized trial, denosumab (a monoclonal IgG2-anti-RANKL-antibody) was investigated as add-on treatment to neoadjuvant chemotherapy (NACT) with two different nab-paclitaxel schedules in early high-risk primary BC. In a translational substudy, we analyzed for the first time the influence of short-term denosumab treatment (24 weeks) on the presence of DTCs. Methods: A total of 167 pts from the GeparX trial were analyzed for DTCs at baseline by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Initially DTC-positive pts were re-analyzed for DTCs after NACT±Denosumab. Results: Overall, 60/167 pts (35.9%) treated with NACT±Denosumab had a pathological complete response (pCR; 55.4% in TNBC, 43.3% in HER2+, 15.3% in HR+/HER2-). At baseline, 43/167 pts (25.7%) were DTC-positive and 41 of those were available for re-analysis of DTCs after NACT±Denosumab. DTC eradication was observed in 77.8% after NACT+Denosumab and in 69.6% after NACT alone (p = 0.726). Due to the limited number of pts eligible for DTC-re-analysis after NACT, a subtype specific analysis for the effect of denosumab was not possible. There was no significant association between pCR and i) the presence of DTCs at baseline (37.1% DTC-negative vs 32.6% positive, p = 0.71) or ii) the eradication of DTCs after NACT±Denosumab (36.7% vs 27.3%, p = 0.72). Notably, in TNBC, we observed a tendency that DTC-positivity at baseline or DTC-persistence after NACT could be associated with reduced pCR rate (40.0% in DTC-positive vs. 66.7% in DTC-negative pts, p = 0.16; 25% in DTC-persistent pts vs. 50% in DTC-eradicated pts, p = 0.59). Conclusions: Denosumab in addition to NACT does not improve pCR, but the suspected effect of denosumab on DTC eradication should be further analyzed in TNBC. Key words: GeparX trial, denosumab, disseminated tumor cells, bone marrow, neoadjuvant chemotherapy. Funding: GeparX and DTC substudy were financially supported by Amgen and Celgene. Clinical trial information: NCT02682693 .

Abstract P3-08-11: The prognostic impact of retinoic acid-induced 2 (RAI2) expression in ERα-positive breast cancer patients

Abstract Background: Breast cancer cells disseminate to the bone marrow and form bone metastases in a large majority of late-stage patients. Retinoic Acid-Induced 2 (RAI2) was reported as a putative suppressor of early hematogenous dissemination of tumor cells to the bone marrow in breast cancer, particularly in estrogen receptor α (ERα)-positive breast cancer. Here, we investigated mRNA expression of RAI-2 in breast cancer patients during long-term follow-up. Materials and methods: A total of 451 invasive breast cancer tissues was available for analysis of RAI2 mRNA using a TaqMan PCR system. We also sought correlations between clinicopathological factors and levels of RAI2 expression in these samples. The expression of markers associated with tumor-initiating capacity, such as SNAI1, SNAI2 and VIM was also analyzed. The median follow-up period was 9.0 years. Survival curves were analyzed using the Kaplan-Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Results: We found positive correlations between low expression of RAI2 mRNA and shorter disease-free survival and overall survival in breast cancer patients (P=0.003, P<0.0001, respectively), which was limited to ERα-positive patients (P=0.04, P=0.0009, respectively), and not seen in ERα-negative patients (P=0.52, P=0.27, respectively). Low RAI2 mRNA levels were positively correlated with high grade, ERα-negativity and PgR negativity. Multivariate analysis indicated that low level RAI2 mRNA expression was an independent factor for survival both overall in breast cancer and in ERα-positive breast cancer patients Multivariate analysis (ERα-positive breast cancer patients) OS Multivariate patientsp valueHR(95%CI)Tumor size≤2cm1390.83691 (Reference) >2cm207 1.08(0.54-2.28)Node statusNegative176<0.00011 (Reference) Positive144 4.72(2.33-10.34)Grade11240.95921 (Reference) 2•3218 0.98(0.50-2.14)RAI2 mRNA expressionhigh140<0.00011 (Reference) middle•low206 4.79(2.14-12.78) . Conclusion:We show that low expression of RAI2 is an independent factor predictive of a poor prognosis in ERα-positive breast cancer patients. RAI2 could be a promising candidate biomarker and therapeutic target in ERα-positive breast cancer to prevent dissemination to the bone marrow. Citation Format: Nishikawa S, Kondo N, Endo Y, Hato Y, Hisada T, Nishimoto M, Dong Y, Okuda K, Kato H, Takahashi S, Nakanishi R, Toyama T. The prognostic impact of retinoic acid-induced 2 (RAI2) expression in ERα-positive breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-08-11.

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

Abstract 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 P1-07-19: Effects of JAK pathway inhibition in pre-clinical models of breast cancer bone marrow metastases

Abstract Bone marrow (BM) metastases are an important problem in metastatic breast cancer. Metastatic-relapse occurs even decades after the first diagnosis and can show different clinical features compared to the primary disease. The cross-talk between cancer cells and the microenvironment influences tumor behaviour and impinges on therapeutic efficiency. To uncover new targets for the inhibition of BM metastases, we performed a transcriptional analysis of BM stromal cells using in-vivo models of breast cancer bone metastases. We identified IL6, and its downstream JAK kinases pathway, as major up-regulated signals in the bone stroma of tumor-bearing mice. Moreover, we found that a significant proportion of BM metastases from breast cancer patient biopsies are positive for pSTAT3 staining, attesting to activation of JAK-STAT signaling. These results provide a rational to investigate the therapeutic potential of a JAK inhibition in bone metastases. We evaluated the effect of two JAK inhibitors: the BSK805, a JAK2 inhibitor, and ruxolitinib a JAK1-JAK2 inhibitor currently in clinical trials for metastatic breast cancer. In pre-clinical models of breast cancer metastases (the triple negative MDA-MB231 SCP1833 and the ER+ EO771), treatment with the JAK inhibitors decreased pSTAT3 signaling in the bone, but surprisingly increased metastastic tumor burden. Considering the importance of the JAK-STAT pathway as a therapeutic target, we are investigating the possible bystander effects of blocking JAK kinases. Analysis of the BM stoma showed that JAK inhibition decreased TRAP+ osteoclasts and affected the mesenchymal compartment, with a significant increase in the BM fat compartment in tumor-bearing animals. The reasons for and the consequences of these changes in the bone stroma are under investigation. As the JAK-STAT signaling pathway is also a key regulator of the hematopoietic compartment, we are studying how JAK inhibitors influence the immune response in tumor bearing mice. Further dissection of the immunomodulatory effect of JAK inhibition and how this influences tumor dissemination and expansion is warranted. Citation Format: Alessia Bottos, Jason Gill, Thomas Radimerski, Alexandar Tzankov, Aleksandra Wodnar-Filipowicz, Nancy E Hynes. Effects of JAK pathway inhibition in pre-clinical models of breast cancer bone marrow metastases [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-19.

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

Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy

The bone marrow (BM) is a major organ of breast cancer (BC) dormancy and a common source of BC resurgence. Gap junctional intercellular communication (GJIC) between BC cells (BCCs) and BM stroma facilitates dormancy. This study reports on a hierarchy of BCCs with the most immature subset (Oct4hi/CD44hi/med/CD24−/+) demonstrating chemoresistance, dormancy, and stem cell properties: self-renewal, serial passaging ability, cycling quiescence, long doubling time, asymmetric division, high metastatic and invasive capability. In vitro and in vivo studies indicated that this subset was responsible for GJIC with BM stroma. Similar BCCs were detected in the blood of patients despite aggressive treatment and in a patient with a relatively large tumor but no lymph node involvement. In brief, these findings identified a novel BCC subset with stem cell properties, with preference for dormancy and in the circulation of patients. The findings establish a working cellular hierarchy of BCCs based on phenotype and functions.

Abstract CN3-1: Detection, clinical relevance, and molecular characteristics of circulating tumor cells

Abstract Early spread of tumor cells is usually undetected even by high-resolution imaging technologies, which prevents effective early intervention. However, sensitive immunocytochemical and molecular assays have become available and enable now the specific detection of 'occult' metastatic tumor cells even at the single-cell stage. These technologies provide the potential to track systemic tumor cell dissemination in cancer patients. Disseminated tumor cells (DTC) have been frequently detected in the bone marrow (BM) of patients with diverse types of solid tumors, suggesting that the BM might be a preferred reservoir for blood-borne DTC. Whether DTC use this environment as niche to persist in a dormant state over many years before they disseminate into other organs or grow out in the BM into overt metastases is subject of current investigations. To understand this stage of dormancy and the conditions enabling DTC to reactivate growth as well as to identify the founder cells of overt metastases (“metastatic stem cells”) are some of the most important and challenging research areas. The observed correlation between DTC in BM and local relapse in breast cancer support the recent experimental findings in mice that these cells might re-circulate back to their primary organ site. If BM is a particular reservoir for DTC, drugs targeting the BM-tumor interaction (e.g., bisphosphonates or antibodies to the RANK ligand) may be efficient to prevent metastatic or even local relapse. In clinical practice, peripheral blood drawings are more acceptable than BM aspirations in solid tumor patients. Many research groups are therefore assessing the clinical value of circulating tumor cells (CTC) for therapy monitoring, which has provided significant prognostic information in metastatic breast cancer, and seems to be superior over conventional imaging methods for response evaluation. The clinical utility of these findings are now being prospectively addressed in a randomized trial SWOG S0500 led by the Southwest Oncology group (www.cancer.gov/clinicaltrials/SWOG-S0500). While the prognostic significance of CTC could be demonstrated for metastatic breast, colorectal or prostate cancer patients, studies on the impact of CTC in early stage cancer patients are still ongoing. Thus far, encouraging results on monitoring of CTC during primary systemic or adjuvant chemotherapy in breast cancer patients were obtained. Phenotypical and molecular characterization of DTC/CTC will be essential to identify new diagnostic and therapeutic targets and contribute to more “tailored” and personalized anti-metastatic therapies (www.dismal-project.eu). The identification of therapeutically relevant targets may contribute to a better selection of cancer patients for targeted therapies. In breast cancer, HER2 has become the most prominent target for biological therapy with a humanized anti-HER2 monoclonal antibody. Currently, all patients are stratified to trastuzumab therapy by primary tumor analysis only. However, recent reports have shown that HER2-positive DTC/CTC can be detected also in patients with HER2-negative primary tumors (Pantel et al NRC 2008), suggesting that additional patients could benefit from HER2-directed therapies. Ongoing clinical studies will reveal whether the HER2 status of CTC may predict response to trastuzumab or other HER2-directed therapies. These results may have also an important impact on the future design of clinical trials testing other promising targets expressed on micrometastatic cancer cells. In conclusion, information obtained from DTC/CTC analyses might be useful to obtain prognostic information, monitor the efficacy of anti-cancer therapies, identify therapeutic targets on metastatic cells and provide new insights into the biology of metastatic progression in cancer patients. Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN3-1

Comparison of the clinical significance of occult tumor cells in blood and bone marrow in breast cancer

Immunocytochemical (ICC) detection of disseminated tumor cells (DTC) in bone marrow (BM) in early breast cancer is an independent prognostic factor. The significance of circulating tumor cells (CTC) in peripheral blood (PB) needs further exploration and comparison to DTC detection. PB and BM were prospectively collected from 341 breast cancer patients median 40 months after operation. PB samples were analyzed for tumor cells by a negative immunomagnetic technique (10x10(6) cells/test). BM aspirates were analyzed by standard ICC (2x10(6) cells/test). CTC were present in 10% of the patients and DTC in 14%. Thirty-seven relapses and 14 breast cancer deaths have occurred at median 66 months after diagnosis. Both CTC-status and DTC-status were significantly associated with disease free survival (DFS) (event rate: CTC-positive 26.5% vs. CTC-negative 9.1%; DTC-positive 29.2% vs. DTC-negative 7.8%) (p<0.001/p<0.001, log rank) and breast cancer specific survival (event rate: CTC-positive 17.6% vs. CTC-negative 2.6%; DTC-positive 12.5% vs. DTC-negative 2.7%) (p<0.001/p<0.001). The presence of both CTC and DTC (n=8) resulted in an especially poor prognosis (p<0.001). In node negative patients, DTC-status, but not CTC-status, predicted differences in DFS (p=0.006 vs. p=0.503). Excluding 23 patients with breast cancer-related events prior to the sample collections, CTC detection was not significantly associated with DFS/distant-DFS (p=0.158/0.193), in contrast to DTC detection (p<0.001/<0.001). Presence of CTC and absence of DTC did not affect DFS (p=0.516). Applied to early stage disease, CTC analysis of increased volumes of PB appears less sensitive and prognostic than standard DTC analysis. Currently, this does not support an exchange of BM with PB for analysis of occult tumor cells.

Detection of Cytokeratin-Positive Cells in Bone Marrow in Breast Cancer and Colorectal Carcinoma in Comparison with Other Factors of Prognosis

A prospective study is presented in which 293 patients suffering from breast cancer and colorectal carcinoma were analyzed for prognostic relevance of detected isolated disseminated tumor cells in bone marrow (IDTBM). The patients underwent surgery in the period from 1995 to 1997 and remained under observation until 1999. The monoclonal antibody A 45-B/B3 was used in the standard immuno-cytochemical method for detecting IDTBM, which represented an independent prognostic factor for survival time in patients with breast cancer or colorectal cancer. In breast cancer, when IDTBM were detected, the survival period was reduced by at least half. When disseminated tumor cells containing the A45-B/B3 antibody were detected in bone marrow, the risk of an earlier relapse of the tumor increased at least fourfold. In colorectal cancer, detection of IDTBM reduced survival time by a factor of 1.2-4.3. The risk of earlier relapse increased when disseminated tumor cells containing the A45-B/B3 antibody were detected in bone marrow by 2.8-8.1. Therefore, the use of IDTBM as an independent prognostic factor would provide an important method for determining the pathological stage of various cancers. Standardization of this technique into a generally accepted method would be especially desirable in treatment of patients with breast or colorectal cancer.