Disseminated Tumor Cells In Bone Marrow Research Articles

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

Abstract 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.

Disseminated tumor cells in pancreatic cancer—an independent prognosticator of disease progression and survival

Pancreatic cancer is one of the most devastating cancers with a 6-month median survival and a 5-year survival rate of 3-5%. Still important aspects of its aggressive biology remain elusive and advanced therapeutic regimens have not been substantially successful. We investigated the prognostic role of disseminated tumor cells (DTC) in bone marrow, a reservoir for early DTC potentially contributing to metastatic progression, of pancreatic cancer patients. After exclusion of patients with different postsurgery diagnosis or missing DTC status (n = 40) a total of 175 patients remained for final analyses. One-hundred and nineteen patients were male and 96 female with a median age of 67 years, 96 patients underwent complete resection. Bone marrow aspirates taken at primary surgery were analyzed for DTC by an immunocytochemical cytokeratin assay and correlated to survival data. Overall 13.7% of patient samples (24/175) harbored DTC in their bone marrow. Histopathological parameters did not correlate significantly. Univariate survival analysis revealed a borderline significant correlation between DTC and decreased progression-free survival (p = 0.069), and was significant for overall survival (p = 0.036). Regarding patients with resected tumors, the respective p-values were 0.058 for progression-free and 0.016 for overall survival. Importantly, the prognostic influence was independent from other risk factors as shown by multivariate analyses for progression-free (p = 0.030, HR: 2.057; CI (95%): 1.073-3.943) and overall survival (p = 0.006, HR: 2.283; CI (95%): 1.260-4.135). The presence of DTC in bone marrow is a strong and independent prognostic factor of survival in patients with pancreatic cancer. Thus, bone-targeting may be a new future therapeutic option for DTC-positive patients.

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis.

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

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

Abstract 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

Mitotic activity and bone marrow micrometastases have independent prognostic value in node positive breast cancer patients

The purpose of this article is to investigate the prognostic value of the mitotic activity index (MAI) and the presence of disseminated tumor cells (DTCs) in bone marrow (BM), in clinically operable breast cancer patients. We compared routinely assessed MAI, classic prognosticators and BM DTCs, detected by a real-time RT-PCR multimarker assay including cytokeratin 19, mammaglobin A and TWIST1 mRNA, in 179 consecutive patients with operable breast cancer. Over a median follow-up of 96 months (range: 1-126 months), 31 (17.3%) patients experienced a systemic relapse and 26 (14.5%) died of breast cancer-related causes. MAI (≥ 10) was strongly associated with breast cancer-related death in lymph node (LN)-negative patients (hazard ratio (HR): 7.0, confidence interval (CI) 1.74-27.9), whereas both BM DTC-status (HR: 3.3, CI 1.25-8.52) and MAI (HR: 3.1, CI 1.08-8.8) were significant in LN-positive patients. With multivariate Cox regression, MAI was the only significant predictor of breast cancer-specific survival (HR 7.0, CI 1.7-27.9) in LN-negative patients. In LN-positive patients, both BM DTC-status and MAI were strong independent predictors of breast cancer-specific survival (HR 3.3, CI 1.25-8.49 and HR 3.1, CI 1.1-8.9), respectively. Where, however, MAI and BM DTC-status as single parameters were replaced by a combination of these, this showed to be the most significant prognostic marker in both LN-negative (HR 7.7, CI 1.2-50) and LN-positive (HR 6.0, CI 1.4 to 26.4) patients with regard to breast cancer-specific survival. A combination of MAI and BM DTC detection identified both LN-negative and LN-positive breast cancer patients with poor prognosis.

Abstract PD04-01: Does Analysis of Disseminated Tumor Cells in Bone Marrow Give Additional Prognostic Information in Primary Breast Cancer? Analysis of Disseminated Tumor Cells in Bone Marrow — Report of a Prospective Study with 5 Years Follow-Up

Abstract Introduction: Disseminated tumor cells (DTC) in bone marrow of patients with breast cancer has, beyond the formerly known and clinically used prognostic markers, in recent publications been identified as an independent predictor of poor prognosis in patients with non-metastatic disease. Detection of DTC could therefore be a way to identify patients with an increased risk of recurrence among patients with a “good” standard prognostic profile. The present prospective study aimed to evaluate the presence and prognostic value of DTC in bone marrow in women undergoing surgery for primary breast cancer. Material and methods: From 1999 to 2003 bone marrow aspirates were obtained from the sternal crest in 569 patients during surgery for primary invasive breast cancer. Tumor cells in bone marrow were identified with cytokeratins (4,5,6,7,8,10,13,14,15,16,18,19) against epithelial cells and analyzed with immunofluorescence and immunohistochemistry. The results were related to clinical follow up for 5 years using distant disease free survival (DDFS) and breast cancer specific survival (BCSS) as end-points by uni-and multivariate analysis including standard prognostic factors. Results: Analysis of DTC were performed in 445 patients and cytokeratin positive cells were found in 159 of these (36%). The presence of DTC in bone marrow was not related to lymph node metastases (N+ 38% vs N-35%, p= 0.4), ER+/ER-(38% vs 30%, p= 0.2) or histological grade (40% grade 1; 37% grade 2; 30% grade 3, p= 0.18). BCSS was related to: lymph node engagement (+ vs -; HR 3.5, p< 0.001), ER (+ vs -; HR 0.55, p= 0.046), histological grade (2 vs 1: HR 4.9, p= 0.03; 3 vs 1: HR 7.4, p= 0.008), age (HR 1.02/year, p= 0.04) and tumor size (>20 mm vs <20 mm: HR 1.8, p= 0.05) in Cox multivariate analysis. Presence of DTC did not correlate either with DDFS (p= 0.97) or BCSS (p= 0.75). In the subgroup N0/ER-, a significantly higher rate of distant metastases was observed in patients with micrometastases in bone marrow (2/12 vs 0/25; p= 0.04; log rank test) and BCSS in the same subgroup was significantly related to the presence of DTC in bone marrow (2/12 vs 0/25; p= 0.04). Conclusion: Detection of micrometastases in bone marrow in primary breast cancer has previously been shown to be a significant independent predictor of poor prognosis in different studies. In a consecutive cohort including unselected patients we were not able to confirm these results. Our data suggest that it is too early to implement the method of bone marrow aspiration and diagnosis of DTC in clinical routine. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-01.

Disseminated Tumor Cells in Bone Marrow Assessed by TWIST1, Cytokeratin 19, and Mammaglobin A mRNA Predict Clinical Outcome in Operable Breast Cancer Patients

Purpose To investigate the prognostic relevance of disseminated tumor cells (DTCs) in bone marrow (BM) assessed by a multimarker mRNA panel consisting of TWIST1, cytokeratin 19 (CK19) and human mammaglobin A (hMAM) mRNA, in patients with early breast cancer. Patients and Methods TWIST1 (gene name: TWIST1), CK19 (gene name: KRT19), and hMAM (gene name: SCGB2A2) mRNA was quantitated in BM samples from 191 operable breast cancer patients by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results Using the highest relative mRNA concentration of TWIST1 in the control population as a cut-off, 5 of the 191 breast cancer patients showed elevated TWIST1 mRNA levels in their BM by real-time RT-PCR. Two of these patients experienced a systemic relapse during a median follow-up of 98 months. Combining these results with previous hMAM and CK19 mRNA quantifications in the same BM samples, 12 (40%) of the 30 patients with BM positive for at least 1 marker (multimarker positive) experienced a systemic relapse as compared with 18 (11%) of the 161 patients with multimarker-negative BMs. The patients with multimarker-positive BM had significantly shorter systemic recurrence-free survival ( P < .001, log-rank test), breast cancer-specific survival ( P < .001), and overall survival ( P = .03). The prognostic relevance of BM multimarker detection appeared to be independent of adjuvant treatment, although the difference was not statistically significant in the subgroup of patients who received adjuvant chemotherapy. Multivariate analysis demonstrated the BM multimarker panel status to be a strong independent predictor of clinical outcome. Conclusion Our results demonstrated the prognostic relevance of BM DTCs assessed by a multimarker mRNA panel consisting of TWIST1, CK19, and hMAM in operable breast cancer patients.

Spontaneous changes in tumour cell dissemination to bone marrow in colorectal cancer

The study aimed to evaluate the incidence of disseminated tumour cells (DTCs) in bone marrow (BM) preoperatively and during follow up and to correlate these with established risk factors in patients with colorectal cancer. We prospectively studied BM in 57 patients using the anti-cytokeratin antibody A45-B/B3. The overall detection rate of DTCs was 23% with a similar detection rate through all stages of the disease. No significant association was found between the presence of DTCs and clinicopathological parameters. After a median follow up of 35.4 months, no differences were found in relapse and overall survival between patients with and without DTC preoperatively. In 31 of 45 patients with local disease, we performed a follow-up BM examination after 1 year. In 26% of the patients, the BM status had changed as compared with the preoperative finding. This is the first study to report the follow up of DTC in BM in colorectal cancer using the A45-B/B3 antibody. The presence of tumour cells in the preoperative BM had no impact on outcome. The BM status had changed after 12 months in a quarter of patients.

Analysis of four human PlGF isoforms and of VEGFR-1 in esophageal carcinoma.

4160 Background: Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor-1 (VEGFR-1) are involved in tumor progression. Humans can express four PlGF isoforms (PlGF1-4), but it is unknown, whether all of them are expressed in cancer. In this retrospective exploratory study, we analyzed expression levels of PlGF1-4 and of VEGFR-1 in primary esophageal tumor tissue and correlated gene expression data with clinical data. We included presence of disseminated tumor cells (DTCs) in the bone marrow in our analysis, which has recently evolved as adverse prognostic factor in different cancers. Methods: Gene expression levels were determined by QRT-PCR in primary tumor samples of 69 patients with esophageal cancer. Subsequently, expression data were correlated with clinical data (pT, pN, pM, DTCs). For analysis of association between expression levels and categorical clinical parameters Wilcoxon's test for independent samples was used. For correlations between quantitative variables, Kendall's tau (τ) was used to measure the degree of dependence. Results: PLGF-1, PLGF-2 and VEGFR-1 were expressed in virtually all analyzed tumor samples. Expression of PLGF-3 and PLGF-4 were limited to 59% and 74% of the analyzed patients, respectively. Expression levels of all PlGF variants were correlated amongst each other (Kendall tau rank>0.55). Expression of PlGF splice variants was not correlated with clinical variables. Interestingly, expression levels of VEGFR-1 predict presence of DTCs in bone marrow (p = 0.002). Patients with DTC in bone marrow had lower expression levels of VEGFR-1 within primary tumor tissue than patients without DTCs (0.004 ± 0.01 vs. 0.07 ± 0.15). In contrast, VEGFR-1 expression level was not correlated with lymphatic or distant macrometastasis, indicating a potential specific role of VEGFR-1 in protecting from dissemination of tumor cells into the bone marrow. Conclusions: All four known isoforms of PLGF are expressed in human esophageal cancer. Furthermore, VEGFR-1 levels are inversely correlated with presence of DTCs in the bone marrow of patients. If validated in prospective studies, VEGFR-1 expression in tumors might serve as novel prognostic biomarker for bone marrow micrometastasis. No significant financial relationships to disclose.

Impact of disseminated tumor cells in bone marrow on outcome of patients with primary ovarian cancer.

e15545 Background: Data concerning the impact of disseminated tumor cells (DTC) in ovarian cancer (EOC) on outcome are still rare. In 109 patients with primary epithelial ovarian cancer, we evaluated whether first-line therapy including radical tumor debulking surgery and chemotherapy with carboplatinum and paclitaxel can impact upon DTC in bone marrow (BM) and analyzed associations of DTC in the BM with clinical data. Methods: DTC were detected in bilateral BM aspirates (10 mL) using density gradient centrifugation and immunocytochemistry, applying the anti-CK antibody A45-B/B3. Disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan-Meier method and statistically evaluated using a two-sided log-rank test. Associations with other clinical data were tested by the chi-squared-test according to Pearson. A significant increase of DTC was assessed if the difference was > = 2 CK+ cells. Results: Before chemotherapy, we identified CK+ cells in 45% (49/109) BM samples with a mean number of 1.9 cells/9x10E6 BM cells (range: 0-37). Puncture after chemotherapy was performed in 67 patients, detecting CK+ cells in 37% (25/67) with a mean number of 3.7 cells/9x10E6 BM (range: 0-100). A significant increase of DTC in the BM was noted in 24% (15/62). In 31% (19/62), chemotherapy eliminated preexisting CK+ cells. In advanced tumor stages FIGOIII/IV, the presence of more than 5 CK+cells before chemotherapy was associated of DTC with less macroscopic complete resection rates in primary surgery (p = 0.042). No other significant associations with clinical data including platinum-sensitivity, grading, tumor stage or the patients' age were observed. FIGO III/IV patients showed a trend for a diminished DFS in patients with a significant increase of DTC in the BM during therapy (estimated means: 24.6 months and 36.2 months, respectively). However, this difference was not statistically significant (p = 0.061). Conclusions: A longer follow-up and greater study population is planned to further evaluate the impact of DTC in ovarian cancer patients. An increase of DTC during therapy might be a prognostic factor in ovarian cancer patients. No significant financial relationships to disclose.

Treg and Teff in the bone marrow of early-stage breast cancer patients with disseminated tumor cells.

e21049 Background: The presence of disseminated tumor cells (DTC) in the bone marrow (BM) is associated with poor survival. The factors regulating appearance and survival of BM DTC are not known. W...

Abstract 841: Comparison of two molecular assays for the detection of circulating tumor cells in blood of primary breast cancer patients and their correlation with disseminated tumor cells in the bone marrow

Abstract Background: In primary breast cancer, the presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor, while the role of circulating tumor cells (CTCs) in blood still has to be proven since a standard method for their detection has not been defined. Molecular approaches are generally more sensitive than cytometric methods and have the additional advantage of a high-throughput technology. Therefore, we examined the presence of CTCs in blood by using two different molecular assays and assessed their correlation with the detection of DTCs in BM of primary breast cancer patients. Patients and Methods: In total, 418 blood samples and 384 BM samples of 377 primary breast cancer patients were studied. Two x 5 ml blood samples were collected and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads. All samples (same cDNAS) were further analyzed by using: a) the AdnaTest BreastCancer (AdnaGen AG) for the detection of three breast cancer associated markers: GA733-2, MUC-1, HER-2 and beta Actin (internal reference gene control), analyzed by capillary electrophoresis on the Agilent Bioanalyzer 2100 and b) real time quantitative RT-PCR for CK-19 mRNA in the Light-Cycler System (Roche Diagnostics). Furthermore, 384 bilateral BM aspirates were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 and DTCs were evaluated with the ARIOL system (Applied Imaging) according to the ISHAGE evaluation criteria. Results: DTCs were found in 115/384 BM samples, resulting in a positivity rate for DTCs of 30%. Applying the AdnaTest, 409/418 samples could be evaluated for the presence of CTCs. 53/409 (13%) samples had detectable CTCs expressing MUC-1 (30%), GA733-2 (25%) and HER2 (70%), respectively. CK19 RT-PCR could be performed in 411/418 samples with a positivity rate of 21% (86/411 samples). A comparison of both molecular CTC assays could be performed in 400 samples with a concordance rate of 71% resulting in 274 blood samples found to be negative by both assays, 11 samples found to be positive by both assays, AdnaTest+/CK19- was documented in 42 cases and AdnaTest-/CK19+ in 73 cases, respectively. Concordant results between the detection of DTCs in the BM and CTCs were found in 65% (AdnaTest) and 62% (CK19 RT-PCR) of all cases, respectively. Discussion: (1) Molecular CTC assays gave different results in 115/400 cases (28.7%) in the same cDNA samples. This could be attributed to the fact that the evaluation of CTCs positivity between these tests is based on a totally different set of genes. (2) Our results might indicate the heterogeneity of CTCs in breast cancer. (3) The low correlation between the detection of DTCs and CTCs by both methods indicates that CTCs may provide prognostic information complementary to DTCs in BM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 841.

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

Insights into minimal residual disease in cancer patients: Implications for anti-cancer therapies

Tumor cell dissemination appears even in patients with small solid tumors, and bone marrow (BM) is a common homing organ for disseminated tumor cells (DTC) derived from various types of primary epithelial tumors. Tumor cells are frequently detected in the BM of cancer patients without any clinical or even histopathological signs of overt metastases. It is crucial, however, to improve and standardize methods for the detection of DTC. The characterization of DTC has shed new light on the process underlying early tumor cell dissemination and metastatic progression in cancer patients. Characterization of DTC should help to identify novel targets for biological therapies aimed at preventing metastatic relapse and to monitor the efficacy of these therapies. Evidence has emerged that the detection of DTC and circulating tumor cells (CTC) in blood may provide important prognostic information and, in addition, might help to monitor the efficacy of therapy. In this article, we summarize the clinical background and the technical aspects of current methods used for the detection and characterization of DTC in BM and CTC in blood, with a special focus on breast cancer.

Prognostic Significance of Real-Time RT-PCR Detection of Disseminated Tumour Cells in Bone Marrow and Circulating Tumour Cells in Patients with Breast Cancer.

Abstract Background: Despite optimal staging and treatment, up to 40% of stage I and II breast cancer (BC) patients will develop recurrent disease over time. These patients are believed to have disseminated tumour cells (DTC) at the time of diagnosis. Detection of minimal disease in bone marrow (BM) has been suggested to be a more direct approach to select metastasis-prone patients among this 'good prognosis group'. Peripheral blood (PB) sampling however is more convenient. The aim of this study was to evaluate whether the detection of DTC in either PB or BM predicts overall survival (OS). The initial analysis was published with a mean follow up time of 786 days (Benoy et al., BrJC 2006). We now report on these data after a mean follow up of nearly 5 years.Material and methods: PB and BM samples were collected from 148 patients with primary (M0, n=116) and metastatic (M+, n=32) BC before the initiation of any local or systemic treatment. PB of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as control group. DTC were detected by measuring relative gene expression (RGE) for cytokeratin 19 (CK19) and mammaglobin (MAM), using a quantitative RT-PCR detection method. The 95 percentile of the RGE for CK19 and MAM of the control group was used as cutoff to determine elevation in BC patients. Kaplan-Meier analysis was used to predict OS.Results: Mean follow up time was 1518 days (+/- 719). Elevated CK19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. MAM expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK19 or MAM expression in BM had a worse OS than patients without elevated expression levels (p=0.002 (CK19) and p=0.001 (MAM)). For PB, no statistical significant difference in OS was observed between patients with or without elevated CK19 (p=0.227), but a strong trend for predicting OS was observed according to MAM status (p=0.054). Separate analyses of M0 and M+ patients revealed only a marked difference in OS according to BM CK19 in the M+ patient group. In M0 patients disease free survival (DFS) was not significantly predicted by CK19 and/or MAM status in BM alone (p=0.173 (CK19), p=0.219 (MAM)), but the presence of the double positive phenotype showed a trend for predicting DFS (p=0.059).Conclusions: DTC measured as elevated CK19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DTC in BM was highly predictive for OS. However, with longer follow up differences in OS between patients with or without elevated CK19 and particularly MAM expression in PB also tend to become more significant. Furthermore, on the long term, double positivity for CK19/MAM in BM seems to predict DFS in patients with localized BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3019.

Abstract CN02-01: Circulating tumor cells as targets for cancer therapy

Abstract Metastatic relapse of carcinoma patients is mainly due to clinically occult micrometastases present at primary diagnosis, but undetectable even by high-resolution imaging technologies. Frequently, traditional prognostic factors are insufficient to predict metastasis and treatment decisions are mainly based on statistical risk parameters. Highly sensitive and specific cytometric and molecular methods enable now the detection of disseminated tumor cells (DTC) in bone marrow (BM) and circulating tumor cells (CTC) in peripheral blood of breast carcinoma patients. The presence of DTC has independent prognostic impact for patients with primary breast cancer with regard to metastatic relapse and overall survival(1) and DTC may even contribute to local relapse (2). Interestingly, bone marrow seems to be a common homing organ for cells derived from various epithelial tumors including breast, prostate, lung and colon cancer (3); (4)). This surprising finding is consistent with recent results obtained in mouse models (5),, supporting the hypothesis that BM might be an important reservoir for metastatic cells from where they can re-circulate into various organs and may be even back to the primary site (6); (7); (8). DTC may have adapted to the special environmental conditions in the BM and may survive in so called “bone marrow niches” over decades. This hypothesis has important clinical implications for the design of future clinical trials with drugs that are able to specifically block the interaction between tumor cells and the bone marrow microenvironement (e.g., bisphosphonate or antibodies to RANK ligand). However, a significant fraction of DTC remain over years in a “dormant” stage, and little is known about the conditions required for the persistence of dormancy or the escape from the dormant phase into the active phase of metastasis formation (9). Transition from a dormant into a dynamic phase may be caused by genetic changes within the disseminated tumor cells (i.e. acquisition of growth factor receptor Her-2/neu amplification (10); (11) but also by the influence of the surrounding bone marrow microenvironment. Furthermore, BM has a particular capability to host stem cells, which may also contribute to keep DTC in a stem cell-like state. This assumption is also supported by the fact that most DTC in BM and blood are in a nonproliferating state and survive systemic chemotherapy (12); (13). Moreover, most DTC in breast cancer patients showed a breast stem cell phenotype (CD44+/CD24- or MUC1-/CK19+) (14); (15). Moreover, epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) - two known stem cell factors - were relevant for the in vitro growth of DTC obtained from BM of cancer patients (16). Nevertheless, strong direct evidence that some of the few DTC or CTC detected in the BM or blood samples have cancer stem cell properties is still missing. Future studies including xenotransplantation of DTC/CTC into immunodeficient mice need to demonstrate that these cells are the actual founder cells of overt metastasis. BM analyses are not well accepted by the medical community for the clinical management patients in breast cancer and other solid tumors. Therefore, most current research efforts are directed to evaluate the clinical utility of CTC detection (17). Because of the high variability of results obtained by different cytometric and molecular approaches, standardization of current technologies is urgently required (18); (19). While the prognostic significance of CTC could be demonstrated for metastatic breast cancer patients (20), studies on the impact of CTC in primary breast cancer patients are still ongoing but the intermediate results are so far promising (21). Moreover, encouraging results on monitoring CTC during primary systemic or adjuvant chemotherapy in breast cancer patients were obtained in recent studies. Further characterization of CTC is pivotal to understand the biology of tumor cell dissemination (22). The molecular characterization of CTC with special emphasis on potential cancer stem cell features and therapeutically relevant targets such as HER2 (23) might improve individual risk assessment and stratification of patients for targeted therapies. This characterization will contribute to more “tailored” and personalized anti-metastatic therapies. At present, the success or failure of anti-cancer therapies is only assessed retrospectively by the absence or presence of overt metastases during the postoperative follow up period. Real-time monitoring of peripheral blood (i.e., during and after systemic adjuvant therapy) for CTC might provide unique information for the clinical management of the individual cancer patient and allow an early change in therapy years before the appearance of overt metastases signals incurability (24). Future clinical trials will show whether the assessment and monitoring of therapeutic targets (e.g., EGF-R, HER2 or VEGF) on CTC (and probably DTC) might become an important diagnostic tool for cancer patients undergoing targeted therapies and may provide new insights into the selection of tumor cells under biological therapies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN02-01.

Minimal residual cancer and its clinical relevance

About one third of axillary node-negative breast cancer patients will develop distant metastases, even if there was no evidence of tumor spread beyond the breast at the time of primary diagnosis. Several studies support the hypothesis that the presence of hematogenous disseminated tumor cells (DTC) in the bone marrow of cancer patients, a condition known as minimal residual disease (MRD), can be regarded as a precursor of clinically manifest distant metastases. MRD can be detected either as DTC in the bone marrow or as circulating tumor cells (CTC) in the peripheral blood. Bone marrow and peripheral blood represent mesenchymal compartments with rare prevalence of epithelial cells without evidence of malignant disease. There are two main approaches for the detection of DTC and/or CTC: 1) immunologic assays using monoclonal antibodies against epithelium-specific proteins, and 2) polymerase chain reaction-based molecular methods detecting tissue-specific transcripts. Although data on the prognostic relevance of DTC in bone marrow are vast, data on the prognostic role of CTC are much less convincing. Sampling peripheral blood instead of bone marrow, however, might enhance the therapeutic implications of detecting MRD in breast cancer, a topic that is examined in this article. This article also examines the major quality criteria for the transfer of new markers into the clinical routine in relation to MRD and reviews whether and in what detail MRD in bone marrow meets these criteria.

Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

IntroductionThe detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.MethodsThe expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1α and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1α or pFAK antibodies, respectively.ResultspFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1α was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1α and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1α and VEGF2.ConclusionsThe expression of pFAK, HIF-1α, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.

Disseminated tumor cells and the risk of locoregional recurrence in nonmetastatic breast cancer

BackgroundIn early breast cancer patients, bone marrow (BM)-disseminated tumor cells (DTCs) were associated with distant metastasis and locoregional recurrence. Our aim was to determine whether BM DTC detection could be related to specific locoregional dissemination of cancer cells, according to radiotherapy volumes. Patients and methodsThe relationship between locoregional recurrence-free survival (LRFS) and DTC detection was evaluated according to the various locoregional volumes irradiated after surgery. ResultsBM DTCs were detected in 94 of 621 stage I–III breast cancer patients (15%) and were not associated with axillary node status. Eighteen patients (2.9%) experienced locoregional recurrence (median follow-up 56 months), of whom eight (44%) were initially BM DTC positive. BM DTC detection was the only prognostic factor for LRFS [P=0.0005, odds ratio=5.2 (2.0–13.1), multivariate analysis]. In BM DTC-positive patients, a longer LRFS was observed in those who were given adjuvant hormone therapy (P=0.03) and radiotherapy to supraclavicular nodes (SCNs)/internal mammary nodes (IMNs) (P=0.055) (multivariate analysis; interaction test: P=0.028). ConclusionsThe presence of DTC in BM may be associated with a different pattern of locoregional cancer cell dissemination and influences LRFS. The possible reseeding of the primary cancer area by DTC could be prevented by systemic hormone therapy but also by SCN/IMN irradiation.