Presence Of Disseminated Tumor Cells Research Articles

The effect of denosumab on disseminated tumor cells (DTCs) of breast cancer patients with neoadjuvant treatment: a GeparX translational substudy

BackgroundDisseminated tumor cells (DTCs) in the bone marrow are observed in about 40% at primary diagnosis of breast cancer and predict poor survival. While anti-resorptive therapy with bisphosphonates was shown to eradicate minimal residue disease in the bone marrow, the effect of denosumab on DTCs, particularly in the neoadjuvant setting, is largely unknown. The recent GeparX clinical trial reported that denosumab, applied as an add-on treatment to nab-paclitaxel based neoadjuvant chemotherapy (NACT), did not improve the patient’s pathologic complete response (pCR) rate. Herein, we analyzed the predictive value of DTCs for the response to NACT and interrogated whether neoadjuvant denosumab treatment may eradicate DTCs in the bone marrow.MethodsA total of 167 patients from the GeparX trial were analyzed for DTCs at baseline by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Initially DTC-positive patients were re-analyzed for DTCs after NACT ± denosumab.ResultsAt baseline, DTCs were observed in 43/167 patients (25.7%) in the total cohort, however their presence did not predict response to nab-paclitaxel based NACT (pCR rates: 37.1% in DTC-negative vs. 32.6% DTC-positive; p = 0.713). Regarding breast cancer subtypes, the presence of DTCs at baseline was numerically associated with response to NACT in TNBC patients (pCR rates: 40.0% in DTC-positive vs. 66.7% in DTC-negative patients; p = 0.16). Overall, denosumab treatment did not significantly increase the given DTC-eradication rate of NACT (NACT: 69.6% DTC-eradication vs. NACT + denosumab: 77.8% DTC-eradication; p = 0.726). In TNBC patients with pCR, a numerical but statistically non-significant increase of DTC-eradication after NACT + denosumab was observed (NACT: 75% DTC-eradication vs. NACT + denosumab: 100% DTC-eradication; p = 1.00).ConclusionThis is the first study worldwide, demonstrating that neoadjuvant add-on denosumab over a short-term period of 24 months does not increase the DTC-eradication rate in breast cancer patients treated with NACT.

Abstract P6-01-43: Predictors of Response to Neoadjuvant Chemotherapy in Breast Cancer: OncotypeDX versus MammaPrint versus Liquid Biopsy

Abstract Background: OncotypeDX (ODX) is a 21-gene recurrence score (RS) assay that is predictive of the benefit of adjuvant chemotherapy in early-stage hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer. MammaPrint (MP) is a 70-gene signature validated to prognosticate distant metastasis and survival. We have previously presented data suggesting that the presence of circulating tumor cells (CTCs) evaluated via liquid biopsy may also have prognostic and predictive utility in HR+/HER2- breast cancer. In this study, we compare the value of ODX, MP and liquid biopsy evaluating CTCs and disseminated tumor cells (DTCs) in predicting pathologic complete response (pCR) following neoadjuvant chemotherapy (NAC). Methods: This retrospective analysis used the National Cancer Database (NCDB) 2004-2017 breast cancer dataset to identify a cohort of patients with HR+/HER2-, AJCC clinical stage I-III breast cancer who received NAC. A series of multiple logistic regression models were used to assess the value of a. ODX (RS < 26 versus ≥26), b. MP, c. the presence of CTCs, and d. the presence of DTCs in predicting pCR to NAC. Each model controlled for age, race, Charlson/Deyo comorbidity scoring, disease histology, grade, and nodal status. Results: A total of n=52,463 patients with stages I-III HR+/HER2- breast cancer received NAC. The patient characteristics of this cohort were as follows: the majority were White (n=42,826, 81.6%), between 50-70 years of age (n=27,683, 52.8%), and with invasive ductal carcinomas of the breast (n=40,197, 76.6%). N=6,111 (11.6%) had Grade I or well-differentiated disease, n=23,546 (44.9%) Grade II or moderately-differentiated disease, and n=2,605 (43.5%) had Grade III or poorly-differentiated disease. N=3,823 have documented recurrence scores based on ODX: with n=2,653 having RS < 26 (69.4%) and n=1,170 (30.6%) having RS ≥26. After controlling for age, race, comorbidity scoring, disease histology, grade and nodal status, RS ≥26 was found to be significantly associated with pCR to NAC (OR 1.85, 95% CI 1.46-2.35, p< 0.001). High-risk scoring per MP was also correlated with pCR but this relationship was not statistically-significant (OR 1.68, 95% CI 0.93-3.03, p=0.084), possibly due to the smaller size of this sample (n=828 patients underwent MP testing). Liquid biopsy data was also limited, with n=250 patients having documented CTC status and n=211 having documented DTC status. Neither the presence of CTCs (OR 0.96, 95% CI 0.44-2.09, p=0.908) nor DTCs (OR 0.61, 95% CI 0.25-1.50, p=0.279) was significantly associated with pCR to NAC. Conclusions: ODX is found to be predictive of pCR to NAC in early-stage, HR+/HER2- breast cancer. Utility of MP and liquid biopsy data in this context appears less robust, however, data is limited. More research is needed to validate existing data in a prospective trial setting, and explore for novel biomarkers across breast cancer subtypes. Citation Format: Nadeem Bilani, Mira Itani, Mohamed Mohanna, Neha Debnath, Barbara Dominguez, Hong Liang, Zeina Nahleh. Predictors of Response to Neoadjuvant Chemotherapy in Breast Cancer: OncotypeDX versus MammaPrint versus Liquid Biopsy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-43.

The prognostic significance of disseminated tumor cells in bone marrow in patients with operable breast cancer: An updated analysis.

e13013 Background: The main cause of death in breast cancer patients is the consequence of the spread and outgrowth of tumor cells at distant sites. The presence of disseminated tumor cells (DTCs) in the bone marrow at diagnosis is considered a predictor for metastasis. Methods: This is an updated analysis of a study in 102 patients with operable breast cancer on the significance of bone marrow micrometastases. The mean follow-up time is 127 months (range 10 - 142, median 129 months). Bone marrow aspirates were analysed for the presence of DTCs by a real time-polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK) and mammaglobin (MAM).The aim of this study is to confirm the association between DTCs and disease-specific survival (DSS) as well as metastasis-free survival (MFS). Results: CK positivity was borderline significant for DSS (p = 0.054). MAM positivity was significantly prognostic for DSS with a hazard ratio (HR) of 3,110 (1,416-6,832) (p = 0,005). Patients positive for both CK and MAM did have a significant worse outcome with a HR for DSS of 7,329 (2,361 – 22,752) (p = 0,001). No significant influence on MFS was identified for CK (p = 0,658) or MAM positivity (p = 0,095). The combination of CK and MAM positivity did however confer a significantly increased risk for MFS (p = 0,043) at a HR of 4,126 (1,409-12,082). Conclusions: This study confirms the role of DTCs as a negative, prognostic factor in patients with operable breast cancer. The combination of CK and MAM is useful to identify this increased risk.

Abstract P2-02-04: In early breast cancer, the ratios of neutrophils, monocyctes and platelets to lymphocytes significantly correlate with the presence of subsets of circulating tumor cells in blood and disseminated tumor cells in the bone marrow

Abstract Background: Circulating tumor cells (CTCs) are the precursors of metastasis and while travelling through the peripheral blood, they crosstalk with different types of blood cells before they finally reach distant organs to settle down as disseminated tumor cells (DTCs). Proinflammatory markers include the neutrophil lymphocyte ratio (NLR), the platelet lymphocyte ratio (PLR) as well as the monocyte lymphocyte ratio (MLR) and the presence of tumor cells as well as specific proinflammatory markers are two independent predictors of worse outcome in breast cancer (BC). However, little is known about the correlation between NLR, PLR, MLR and subsets of CTCs as well as DTCs in early, non-metastatic BC. Here we evaluated the correlation of NLR, PLR, MLR and the presence of epithelial CTCs (eCTCs), CTCs in epithelial mesenchymal transition (EMT-CTCs) as well as DTCs to better identify patients at risk, to monitor treatment reponse and probably adjust therapeutic options. Methods: The counts of peripheral neutrophils, lymphocytes, monocytes and platelets to determine NLR, PLR and MLR as well as clinicopathological data during diagnosis were retrospectively recorded for 171 patients (pts) diagnosed with BC from July 2006 to December 2012, before the start of therapy. NLR, PLR and MLR were calculated from peripheral blood cell counts and their optimal cutoff levels were determined by the 75% percentile resulting in the following values: NLR: 3.13, MLR: 0.39 and PLR: 222.3, respectively. 170/171 pts were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibodyA45-B/B3 (Clodronate intake was recommended in case of DTC-positivity). CTCs were determined in 155/171 pts applying positive immunomagnetic selection using the AdnaTest BreastCancerSelect. The recovered cDNA was tested for the presence of eCTCs and EMT-CTCs using the AdnaTests BreastCancerDetect and EMTDetect. Pts were considered CTC-positive if at least one of the two tests were positive. Statistical analysis included descriptive reporting, U-test for group differences, Rho correlations for dependencies and Fisher's chi-square test for distributional differences. Rho correlations were used to assess the relationship between NLR, PLR, MLR and tumor cells as well as progression-free survival (PFS) and overall survival (OS). Results: DTCs were detected in 34% of the pts and at least one CTC-subtype was found in 28% of the pts. Whereas the presence of DTCs was not associated with PFS or OS, the presence of CTCs significantly correlated with a shorter PFS (p=0.046) and OS (p=0.018). However, neither eCTCs nor EMT-CTSs alone were of prognostic significance. Enhanced lymphocyte (p=0.025) and monocyte counts (p=0.039) as well as a low PLR (p=0.032) significantly correlated with a reduced PFS in pts still alive whereas an enhanced MLR showed a high correlation with a shorter PFS (p=0.007) and OS (p=0.021) in deceased pts. Whereas MLR significantly correlated with the presence of EMT-CTCs (p=0.045), the presence of eCTCs significantly associated with an elevated NLR (p=0.003) and enhanced lymphocyte (p=0.007) as well as monocyte counts (p=0.012). No significant correlations were found for NLR, PLR and MLR with DTCs, however, DTC-positive pts, harboring a lower PLR, had a significant shorter OS (p=0.043). Conclusion: Here we show that proinflammatory markers in blood are closely related to the presence of different CTC subsets in early BC while no direct correlation was found for tumor cell spread to the bone. These findings might improve the prognostication of these pts and probably help to monitor response to therapy and adjust treatment options. Citation Format: Sabine Kasimir-Bauer, Ebru Karaaslan, Olaf Hars, Ann-Kathrin Bittner, Oliver Hoffmann, Rainer Kimmig. In early breast cancer, the ratios of neutrophils, monocyctes and platelets to lymphocytes significantly correlate with the presence of subsets of circulating tumor cells in blood and disseminated tumor cells in the bone marrow [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-04.

Systematic Evaluation of HLA-G 3'Untranslated Region Variants in Locally Advanced, Non-Metastatic Breast Cancer Patients: UTR-1, 2 or UTR-4 are Predictors for Therapy and Disease Outcome.

Despite major improvements in diagnostics and therapy in early as well as in locally advanced breast cancer (LABC), metastatic relapse occurs in about 20% of patients, often explained by early micro-metastatic spread into bone marrow by disseminated tumor cells (DTC). Although neoadjuvant chemotherapy (NACT) has been a successful tool to improve overall survival (OS), there is growing evidence that various environmental factors like the non-classical human leukocyte antigen-G (HLA-G) promotes cancer invasiveness and metastatic progression. HLA-G expression is associated with regulatory elements targeting certain single-nucleotide polymorphisms (SNP) in the HLA-G 3’ untranslated region (UTR), which arrange as haplotypes. Here, we systematically evaluated the impact of HLA-G 3’UTR polymorphisms on disease status, on the presence of DTC, on soluble HLA-G levels, and on therapy and disease outcome in non-metastatic LABC patients. Although haplotype frequencies were similar in patients (n = 142) and controls (n = 204), univariate analysis revealed that the UTR-7 haplotype was related to patients with low tumor burden, whereas UTR-4 was associated with tumor sizes >T1. Furthermore, UTR-4 was associated with the presence of DTC, but UTR-3 and UTR-7 were related to absence of DTC. Additionally, increased levels of soluble HLA-G molecules were found in patients carrying UTR-7. Regarding therapy and disease outcome, univariate and multivariate analysis highlighted UTR-1 or UTR-2 as a prognostic parameter indicative for a beneficial course of disease in terms of complete response towards NACT or progression-free survival (PFS). At variance, UTR-4 was an independent risk factor for a reduced OS besides already known parameters. Taken into account the most common HLA-G 3’UTR haplotypes (UTR-1–UTR-7, UTR-18), deduction of the UTR-1/2/4 haplotypes to specific SNPs revealed that the +3003C variant, unique for UTR-4, seemed to favor a detrimental disease outcome, while the +3187G and +3196G variants, unique for UTR-1 or UTR-2, were prognostic parameters for a beneficial course of disease. In conclusion, these data suggest that the HLA-G 3’UTR variants +3003C, +3187G, and +3196G are promising candidates for the prediction of therapy and disease outcome in LABC patients.

Detection of disseminated tumor cells and their relationship with a population of bone marrow lymphocytes in patients with non-small cell lung cancer

Introduction.Detection of disseminated tumor cells (DTC) in solid tumors is an important component of the assessment of disease prognosis. Bone marrow damage is common. There is evidence indicating an important role for bone marrow lymphocyte subpopulations in hematogenous metastasis.
 Aim.To evaluate the frequency of bone marrow damage in patients with non-small cell lung cancer (NSCLC) based on the detection of DTC by flow cytometry, as well as their effect on the population of bone marrow lymphocytes.
 Materials and methods.62 bone marrow samples of patients with a verified diagnosis of NSCLC: adenocarcinoma (33), squamous cell carcinoma (27), other types (2). Methods: morphological, multicolor flow cytometry. Studied DTC, lymphocyte populations CD3, CD4, CD8, CD19, CD20, CD16, CD27. Collection and analysis: FACS Canto II, USA, Kaluza Analysis v2.1.
 Results.In bone marrow, DTC (EPCAM+CD45-) were found in 43.5% of patients with NSCLC (1 cell per 10 million myelocariоcytes was taken as the threshold value). The presence of DTC did not correlate with the size of the tumor, the status of the lymph nodes, and the stage of the tumor process. DTC was more often observed in more differentiated tumors (p=0.023). A significant increase in the level of subpopulations of CD16+CD4-NK-cells (p=0.002), CD27+CD3+T-cells (p=0.015) with bone marrow damage was revealed.
 Conclusion.The possibility of detecting DTC in the bone marrow of patients with NSCLC was established, in 43.5% of patients with NSCLC in the bone marrow DTC was detected, and their presence was established even with a localized tumor process. More frequent bone marrow damage was observed with well-differentiated tumors. The relationship between DTC and bone marrow lymphocyte populations was revealed: subpopulations of CD16+CD4-, CD27+CD3+.

Cytokeratin-positive cells in the bone marrow from patients with pancreatic, periampullary malignancy and benign pancreatic disease show no prognostic information

BackgroundPancreatic and periampullary carcinoma are aggressive tumours where preoperative assessment is challenging. Disseminated tumour cells (DTC) in the bone marrow (BM) are associated with impaired prognosis in a variety of epithelial cancers. In a cohort of patients with presumed resectable pancreatic and periampullary carcinoma, we evaluated the frequency and the potential prognostic impact of the preoperative presence of DTC, defined as cytokeratin-positive cells detected by immunocytochemistry (ICC).MethodsPreoperative BM samples from 242 patients selected for surgical resection of presumed resectable pancreatic and periampullary carcinoma from 09/2009 to 12/2014, were analysed for presence of CK-positive cells by ICC. The median observation time was 21.5 months. Overall survival (OS) and disease-free survival (DFS) were calculated by Kaplan-Meier and Cox regression analysis.ResultsSuccessful resections of malignant tumours were performed in 179 of the cases, 30 patients resected had benign pancreatic disease based on postoperative histology, and 33 were deemed inoperable intraoperatively due to advanced disease. Overall survival for patients with resected carcinoma was 21.1 months (95% CI: 18.0–24.1), for those with benign disease OS was 101 months (95% CI: 69.4–132) and for those with advanced disease OS was 8.8 months (95% CI: 4.3–13.3). The proportion of patients with detected CK-positive cells was 6/168 (3.6%) in resected malignant cases, 2/31 (6.5%) in advanced disease and 4/29 (13.8%) in benign disease. The presence of CK-positive cells was not correlated to OS or DFS, neither in the entire cohort nor in the subgroup negative for circulating tumour cells (CTC).ConclusionsThe results indicate that CK-positive cells may be present in both patients with malignant and benign diseases of the pancreas. Detection of CK-positive cells was not associated with differences in prognosis for the entire cohort or any of the subgroups analysed.Trial registrationclinicaltrials.gov (NCT01919151).

DETECTION OF DISSEMINATED TUMOR CELLS IN THE BONE MARROW OF PATIENTS WITH NON-SMALL CELL LUNG CANCER

Introduction. There is evidence that disseminated tumor cells (DOCs) in the bone marrow (BM) are precursors of subsequent distant metastases. The detection of DOCs in non-small cell lung cancer (NSCLC) will provide important information about the features of metastasis, as well as the possibilities of identifying new targets for the treatment of NSCLC. Purpose of the study is to evaluate the possibility of detection DOCs in BM and to identify the frequency of BM involvement in patients with NSCLC. Materials and methods . Morphological and immunological methods studied 62 BM samples from patients with NSCLC. DOCs analysis was performed using flow cytometry (FACSCanto II, USA, Kaluza Analysis v 2.1 software), monoclonal antibodies to CD45, cytokeratins directly labeled with various fluorochromes were used. Results . DOCs (EPCAM+CD45–) in the BM were found in 43.5 % of patients (threshold level: 1 cell per 10 million myelocaricytes). The presence of DOCs did not correlate with tumor size, lymph node status, stage of the tumor process. The highest detection rates of DOCs were observed at stages IA and IIA: 60.7 % (3/5) and 58.3 % (7/12) respectively. BM involvement in adenocarcinoma was observed in 45 % (15/33) cases, in squamous cell carcinoma – in 37 % (10/27) samples (p = 0.501). It was found that DOCs are more often detected in more differentiated tumors (p = 0.023). Significant correlations between the presence of DOCs in the BM and myelogram parameters have not been established. A decrease in the number of granulocyte germ cells was observed in 4 % of BM involvement (p = 0.036). Conclusion. The possibility of detecting DOCs in the BM of NSCLC patients has been established. BM involvement was 43.5 %. DOCs are detected even in the early stages of NSCLC. Relationship between BM involvement and the degree of tumor differentiation was found. More frequent BM involvement was observed in adenocarcinoma compared with squamous cell carcinoma of the lung. Compliance with patient rights and principles of bioethics The study protocol was approved by the biomedical ethics committee of N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russia. All patients gave written informed consent to participate in the study.

Comparative Analysis of Blood and Bone Marrow for the Detection of Circulating and Disseminated Tumor Cells and Their Prognostic and Predictive Value in Esophageal Cancer Patients.

Hematogenic tumor cell spread is a key event in metastasis. However, the clinical significance of circulating tumor cells (CTC) in the blood and disseminated tumor cells (DTC) in bone marrow is still not fully understood. Here, the presence of DTC and CTC in esophageal cancer (EC) patients and its correlation with clinical parameters was investigated to evaluate the CTC/DTC prognostic value in EC. This study included 77 EC patients with complete surgical tumor resection. CTC and DTC were analyzed in blood and bone marrow using nested CK20 reverse transcription-nested polymerase chain reaction (RT-PCR) and findings were correlated with clinical data. Twenty-seven of 76 patients (36.5%) showed CK20 positivity in the blood, 19 of 61 patients (31.1%) in bone marrow, and 40 (51.9%) of 77 patients were positive in either blood or bone marrow or both. In multivariate analyses, only the DTC status emerged as independent predictor of overall and tumor specific survival. Our study revealed that, while the presence of CTC in blood is not associated with a worse prognosis, DTC detection in the bone marrow is a highly specific and independent prognostic marker in EC patients. Larger cohort studies could unravel how this finding can be translated into improved therapy management in EC.

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 .

Impact of disseminated tumor cells in the bone marrow on survival and disease progression in patients with left-sided colorectal cancer.

Disseminated tumor cells (DTCs) are a subset of circulating tumor cells that migrate to the bone marrow. Colorectal cancer is a heterogeneous disease depending on the site of the primary tumor. We aimed to assess the association between the presence of DTCs in the bone marrow and tumor characteristics as well as long‑term treatment outcomes in patients with left‑sided colorectal cancer. This prospective study included 91 patients with left‑sided colorectal cancer (37 with colon cancer and 54 with rectal cancer) treated between 2007 and 2012 in a single tertiary center. Fifteen patients had stage I cancer; 26, stage II; 26, stage III; and 24, stage IV. Overall survival and cancer relapse rates were compared between patients with different cancer stages and DTC status. Bone marrow DTCs were identified in 42 patients (46.1%). The prevalence of DTCs was not related to tumor infiltration depth, nodal involvement, distant metastasis, tumor stage, or primary tumor site. The 5‑year overall survival rates were 59.5% and 53% in the DTC‑positive and DTC‑negative groups, respectively (P = 0.19). There was a notable trend favoring survival in patients with DTCs with stage II and III disease (both separately and when combined). The number of metachronous distant metastases was significantly lower in DTC‑positive patients. The presence of DTCs in the bone marrow is not associated with primary tumor characteristics and seems to reduce metastasis formation in left‑sided colorectal cancer. There is also a trend for improved overall survival in DTC‑positive patients. These results are intriguing and warrant further confirmation.

Dissimilar patterns of tumor-infiltrating immune cells at the invasive tumor front and tumor center are associated with response to neoadjuvant chemotherapy in primary breast cancer

BackgroundTumor-infiltrating lymphocytes (TILs) are described as an important immune modulator in the tumor microenvironment and are associated with breast cancer (BC) outcome. The spatial analysis of TILs and TIL subtype distribution at the invasive tumor front (ITF) and the tumor center (TC) might provide further insights into tumor progression.MethodsWe analyzed core biopsies from 87 pre-therapeutic BC patients for total TILs and the following subtypes: CD3+, CD4+, CD8+, CD20+ and CD68+ cells in correlation to clinicopathological parameters and disseminated tumor cells (DTCs) in the bone marrow.ResultsTILs and TIL subtypes showed significantly different spatial distribution among both tumor areas. TILs, especially CD3+ T cells were associated with the tumor status and tumor grading. BC patients responding to neoadjuvant chemotherapy had significantly more TILs and CD3+ T cells at the TC. The presence of DTCs after NACT was related to CD4+ infiltration at the TC.ConclusionThe dissimilar spatial association of TILs and TIL subtypes with clinicopathological parameters, NACT response and minimal residual disease underlines the necessity of detailed TIL analysis for a better understanding of immune modulatory processes.

Abstract B070: Epithelial-marker absent and disease-marker specific circulating and disseminated tumor cells are rarely detected in men with localized prostate cancer

Abstract Introduction and Objectives: Epithelial-marker negative circulating tumor cells (CTCs) have recently been discovered in metastatic prostate cancer (PCa). Many popular CTC detection methods, including the AdnaTest, utilize a selection-based first step of bead pull down against an epithelial marker such as EpCAM. It has previously been shown that disseminated tumor cells (DTCs) are rare at the time of localized PCa using the AdnaTest. Here we queried the matched epithelial-marker depleted buffy coat (DBC) fraction of the AdnaTest for the presence of DTCs and CTCs in a cohort of localized patients undergoing radical prostatectomy (RP). Methods: 8 mL of bone marrow (BM) were collected from 28 patients (5 controls, 2 metastatic, and 21 localized) along with 8 mL of matched peripheral blood (PB) from 11 patients (3 controls, 2 metastatic, 6 localized). 5 mL of BM and PB were evaluated with the AdnaTest ProstateCancer Select kit (Qiagen) to enrich for EpCAM positive cells using immunomagnetic beads. The DBCs having the remaining cells after AdnaTest enrichment were isolated and queried for the presence of DTCs and CTCs. Cells were lysed and reverse transcribed into cDNA followed by real-time qPCR for the expression of the prostate-specific markers, NKX3.1, androgen receptor (AR), prostate specific antigen (PSA), and HOXB13, as well as the epithelial marker EpCAM. The buffy coats from the remaining 3 mL of nonenriched BM and PB were isolated and stored in -80°C for future evaluation. Results: EpCAM, NKX3.1, and AR were expressed in localized, control, and metastatic BM and PB and thus were nonspecific to identifying prostate CTCs and DTCs. PSA and HOXB13 positive cells were only identified in the metastatic setting. With the AdnaTest, PSA and HOXB13 positive DTCs were detected in 0/21 (0%) localized patients, but were identified in 2/2 (100%) metastatic samples. Positive PSA and HOXB13 matched in all cases. No controls expressed PSA or HOXB13. The DBC fraction was contaminated with EpCAM positive cells in 27/28 (96%) BM samples in addition to 8/11 (73%) PB specimens. PSA or HOXB13 expression were not detected in any DBC, demonstrating the absence of epithelial-marker negative DTCs. In two instances, EpCAM was detected in the PB depleted fraction but not with the AdnaTest. Conclusion: EpCAM, NKX3.1, and AR are nonspecific, while PSA and HOXB13 are prostate-specific markers in the PB and BM. PSA and HOXB13 positive prostate CTCs and DTCs are rarely identified in localized men undergoing RP. Epithelial-marker negative DTCs and CTCs were not detected in localized men. The AdnaTest bead selection step did not capture all epithelial cells, and ongoing investigation into a selection-free CTC/DTC detection platform is necessary. Citation Format: Stephanie Glavaris, Heather Chalfin, Changxue Lu, Yan Chen, Emily Caruso, Kenneth Valkenburg, Jun Luo, Kenneth Pienta. Epithelial-marker absent and disease-marker specific circulating and disseminated tumor cells are rarely detected in men with localized prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B070.

Abstract 5118: Development of new alternative in vivo models to study early metastasic dissemination in breast cancer

Abstract Breast cancer is the first cause of death from female cancer. After the treatments against the primary tumor, a few cells can prevail in a state of quiescence for a long period of time, becoming minimal residual disease. There is a clear need for alternative techniques to enable an early detection of the disease for a better understanding of it and the generation of therapies capable of acting with a smaller tumor burden. Although many models have been developed for the detection of macrometastasis, there is a limitation of in vivo models that allow the identification of disseminated cells, micrometastasis, or analyze another steps of the metastasic cascade, as embolization or cell arrest. The aim of this work is the generation of in vivo models to enable the detection of disseminated tumor cells (DTCs) We began our study by evaluating the presence of disseminated tumor cells in three in vivo models and quantification by FACS. To this end, we generated the F3II murine triple negative mammary carcinoma line expressing the fluorescent protein GFP (F3II GFP); the F3II line was used as control. In s.c heterotopic model, we observed the presence of positive cells in tumors and livers of mice inoculated with F3II GFP (Student's t test) 30 days post inoculation. In the s.c orthotopic model, we detected positive cells in tumors of mice inoculated with F3II GFP (Student's t test). In both models, we did not observe significant differences in the presence of lung DTCs. In experimental metastasis models, we detected DTCs 15 days post inoculation in lungs of mice injected i.v with F3II GFP (Mann Whitney test).We carried out the search for cells disseminated at 24 h in order to obtain an in vivo model that evaluates the establishment capacity of these cells, and we observed that the percentage of GFP positive cells was higher in those mice inoculated iv with F3II GFP (Student´s t test). In addition, the presence of DTCs was detected by fluorescence microscopy in blood smears of these mice, suggesting that at 24 h there are fluorescent tumor cells in both lung and blood. In conclusion, although the established models allowed us to detect DTCs, the experimental metastasis model was more efficient in the detection and quantification of disseminated tumor cells. These models allows the detection and quantification of DTCs in the lung and blood circulation within 24 hours and 15 days after inoculation and could be used to study the mechanisms of metastasic dissemination involved in these cells and to test the capacity of various anti-tumor drugs to the early stage of metastasis Citation Format: Johanna Elena Sidabra, Carla Sabrina Capobianco, Maria Florencia Gottardo, Daniel Fernando Alonso, Hernan Gabriel Farina. Development of new alternative in vivo models to study early metastasic dissemination in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5118.

A high-risk 70-gene signature is not associated with the detection of tumor cell dissemination to the bone marrow.

The 70-gene signature (70-GS) is a prognostic tool, grouping patients in risk groups to assess their need for adjuvant chemotherapy. Tumor cell dissemination to the bone marrow is a marker of minimal residual disease and associated with impaired survival. In this study, we aimed to evaluate whether 70-GS is associated with the presence of disseminated tumor cells (DTCs) in the bone marrow of patients with early breast cancer. In patients with hormone receptor-positive HER2-negative early breast cancer, the 70-GS was obtained and the presence of DTCs was immunohistochemically evaluated using cytokeratin staining with the A45-B/B3 antibody. 149 patients were included into the analysis. 40 (27%) had a high-risk 70-GS and 35 (23%) had detectable DTCs in their bone marrow. 9 (22%) of the 40 patients with high-risk 70-GS and 26 (24%) of the 109 patients with a low-risk 70-GS were positive for DTCs (p=0.863). As both 70-GS and DTC detection are known prognostic factors but do not seem to correlate, a follow-up on a larger cohort is warranted to evaluate if a combination of the two is able to better stratify the relapse risk in early breast cancer patients.

Disseminated tumor cells are not associated with established risk factors, L1CAM immunoreactivity and outcome in endometrial carcinoma.

The presence of disseminated tumor cells (DTC) in the bone marrow of endometrial carcinoma patients has been demonstrated previously. In contrast to breast cancer, no prognostic significance or association with clinicopathological features was revealed for endometrial carcinoma so far. The aim of this study was to investigate DTC in a large patient cohort with in-depth pathology review data available and to study DTC occurrence in the context of L1CAM and long-term disease specific follow-up. Patients treated for endometrial carcinoma at the Tuebingen University Women's hospital between 2003 and 2013 were identified. Cases with previous expert central pathology review including L1CAM immunohistochemistry and bone marrow aspirates available were selected. The presence of DTC and L1CAM expression was studied immunohistochemically. In 395 cases with a confirmed diagnosis of endometrial carcinoma, bone marrow aspirates were available. DTC were detected in 17.2%. The presence of DTC was independent from tumor histology, grade, lymphovascular space involvement (LVSI), FIGO stage, myoinvasion, L1CAM immunoreactivity, and nodal metastasis. DTC occurred less frequently in cases with a microcystic elongated and fragmented (MELF) pattern of invasion (2.2 vs. 21.8%, p=0.0003). Disease progression was distributed equally among patients with and without DTC present. We were able to confirm previous findings of DTC presence in a large well-characterized cohort of endometrial carcinoma patients. DTC are detectable in almost one-fifth of endometrial carcinoma and occur less frequently with a MELF pattern of invasion. Further studies investigating the role of DTC in endometrial carcinoma are warranted.

Are disseminated tumor cells in bone marrow and tumor-stroma ratio clinically applicable for patients undergoing surgical resection of primary colorectal cancer? The Leiden MRD study.

PurposeCurrent TNM staging does not appropriately identify high-risk colorectal cancer (CRC) patients. The aim of this study was to evaluate whether the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) and the presence of stroma in the primary tumor, i.e., the tumor-stroma ratio (TSR), in patients undergoing surgical resection of primary CRC provides information relevant for disease outcome.MethodsPatients with primary CRC (n = 125), consecutively admitted for curative resection between 2001 and 2007, were included in the study. All patients underwent BM aspiration before surgery. Detection of tumor cells was performed using immunocytochemical staining for cytokeratin (CK-ICC). The TSR was determined on diagnostic H&E stained sections of primary tumors.ResultsDTCs were detected in the BM of 23/125 patients (18 %). No association was found between BM status and overall survival (HR 0.97 (95 % CI 0.45–2.09), p = 0.93). Also, no significant difference was found in their 5-year survival rate (resp. 72 % and 68 % for BM-positive versus BM-negative patients). The TSR was found to be associated with a worse overall survival (HR 2.16, 95 % CI 1.02–4.57, p = 0.04) with 5-year survival rates of 84 % versus 62 % for stroma-low and stroma-high patients, respectively. No relation was found between the presence of DTCs and TSR.ConclusionsOur data indicate that the presence of DTCs in the BM of CRC patients is not associated with disease outcome. The TSR was, however, found to be associated with a worse overall survival, which indicates that for CRC the tumor microenvironment plays an important role in its behavior and prognosis.

F06 - Integrin a5 is an independent prognosis factor and a potential therapeutic target for breast cancer bone metastasis

Background: Using an extensive bioinformatic approach we identified integrin &agr;5 subunit as a novel potential target to treat bone dissemination from breast cancer. Aim of this study is to confirm the value of this target Material and methods: Integrin &agr;5 mRNA expression was quantified by qRT-PCR in 427 breast cancer patients. &agr;5 protein levels on primary tumor was correlated with presence of disseminated tumor cells (DTCs) in bone marrow at the time of surgery in further 268 breast cancer patients. BALB/c mice were injected with human MDA-MB-231 &agr;5 silenced or scramble. Animals were killed on day 14 after tumor cell inoculation; lung and bone marrow were flushed from the hind limbs for DTC colony assay. Alternatively mice were treated with an anti a5 moAb (M200) or vehicle. Treatment was performed 3 times per week starting the day before intra-arterial inoculation with luciferase expressing human MDA-MB-231/B02 breast cancer cells, which selectively metastasize to bone. In an additional protocol, animals were culled on day 7 after tumor cell inoculation, and the bone marrow was flushed for DTC colony assay. Results: An high a5 expression was associated with shorter bone metastasis-free survival, both in univariate (P = 0.024) and multivariate analysis (P = 0.04). Moreover we found significant (P = 0.039) positive association between a5 protein expression on primary cancer and presence of DTCs in bone marrow. Taking advantage of the ability of the MDA-MB-231 cancer cell to metastatize both in bone and lung, we found that abrogating a5 gene function dramatically reduced the number of bone micrometastases (P = 0.015) without affecting lung dissemination. In addition treatment of MDA-MB-231/B02 injected animals with M200 antibody significantly delayed the onset of skeletal lesions (P = 0.02) and caused a 50% reduction in the extent of osteolytic lesions (P = 0.038), compared to vehicle. This difference was accompanied with a sharp reduction of tumor burden (P = 0.02), as determined by bioluminescence. Histomorphometric analysis of metastatic legs showed that M200 treatment decreased skeletal tumor burden (P = 0.027) and increased the bone volume (P = 0.02), compared to vehicle. Additionally, the number of DTC colonies from M200-treated mice was dramatically decreased compared with vehicle (P< 0.001). Conclusions: Our results suggest that &agr;5 integrin expression in breast cancer cells facilitates bone marrow micrometastasis formation and the subsequent development of osteolytic lesions.

Detection of disseminated tumor cells in lymph nodes from patients with early stage non-small cell lung cancer.

BackgroundThe regional lymph node involvement is a major prognostic factor in patients with non-small cell lung cancer (NSCLC) undergoing surgical resection. Disease relapse is common, suggesting that early disseminated disease is already present in the regional lymph nodes at the time of surgery, and that the current nodal staging classification might be suboptimal. Early detection of disseminated tumor cells (DTCs) in lymph nodes could potentially enable identification of subcategories of patients with high risk of disease relapse.MethodLymph node samples were collected from 128 NSCLC patients at the time of surgery and the presence of DTCs determined by immunomagnetic selection (IMS) using the MOC31 antibody recognizing EpCAM. Results obtained with IMS were compared to the pathological staging obtained by histopathology. Associations between the presence of DTCs and clinicopathological variables and patient outcome were investigated.ResultsDTCs were detected in 40 % of the lymph node samples by IMS. Their presence was significantly associated with pN status as assessed by histopathology, and samples from 83 % of the patients with lymph node metastases (pN1-2) had detectable DTCs. In the group of patients who were negative for lymph node metastases by standard histopathology (pN0) DTCs were detected in 32 %. The presence of DTCs was not associated with any other clinicopathological variables. Patients with IMS-positive samples showed decreased relapse free survival compared to patients with IMS-negative samples, but the difference was not statistically significant. The pN status was significantly associated with both relapse free and overall survival, but the presence of DTCs had no prognostic impact in the subcategory of patients with pN0 status.ConclusionOur findings do not support further development of lymph node DTC detection for clinical use in early stage NSCLC.

Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells.

BackgroundDisseminated tumor cells (DTCs) can be detected using ultrasensitive immunocytochemical assays and their presence in the bone marrow can predict the subsequent occurrence of overt metastasis formation and metastatic relapse. Using expression profiling on early stage primary breast tumors, low IRX2 expression was previously shown to be associated with the presence of DTCs in the bone marrow, suggesting a possible role of IRX2 in the early steps of metastasis formation. The purpose of this study is to gain insights into the significance of IRX2 protein function in the progression of breast cancer.MethodsTo assess the physiological relevance of IRX2 in breast cancer, we evaluated IRX2 expression in a large breast cancer cohort (n = 1992). Additionally, constitutive IRX2 over expression was established in BT-549 and Hs578T breast cancer cell lines. Subsequently we analyzed whether IRX2 overexpression effects chemokine secretion and cellular motility of these cells.ResultsLow IRX2 mRNA expression was found to correlate with high tumor grade, positive lymph node status, negative hormone receptor status, and basal type of primary breast tumors. Also in cell lines low IRX2 expression was associated with mainly basal breast cancer cell lines. The functional studies show that overexpression of the IRX2 transcription factor in basal cell lines suppressed secretion of the pro-metastatic chemokines and inhibited cellular motility but did not influence cell proliferation.ConclusionOur results imply that the IRX2 transcription factor might represent a novel metastasis associated protein that acts as a negative regulator of cellular motility and as a repressor of chemokine expression. Loss of IRX2 expression could therefore contribute to early hematogenous dissemination of breast cancer by sustaining chemokine secretion and enabling mobilization of tumor cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1907-4) contains supplementary material, which is available to authorized users.