Detection Of Disseminated Tumor Cells Research Articles

Aprospective study of detection and clinical significance of bone marrow tumor cells in small cell lung cancer

Objective: To investigate the detection of bone marrow tumor cells in small cell lung cancer (SCLC) patients and their relationship with clinical features, treatment response and prognosis. Methods: A total of 113patients with newly diagnosed SCLC from January 2018 to October 2022 at Beijing Chest Hospital were prospectively enrolled. Before treatment, bone marrow was aspirated and separately submitted for tumor cells detection by liquid-based cytology and disseminated tumor cells (DTCs) detection by the substrction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) platform. The correlation between the detection results of the two methods with patients' clinical features and treatment response was evaluated by Chi-square. Kaplan-Meier method was applied to create survival curves and the Cox regression model was used for multivariate analysis. Results: The positive rate of bone marrow liquid-based cytology in SCLC was 15.93% (18/113). The liver and bone metastases rates were significantly higher (55.56% vs 11.58% for liver metastasis, P＜0.001; 77.78% vs 16.84% for bone metastasis, P＜0.001) and thrombocytopenia was more common (16.67% vs 2.11%, P=0.033) in patients with tumor cells detected in liquid-based cytology than those without detected tumor cells. As for SE-iFISH, DTCs were detected in 92.92% of patients (105/113), the liver and bone metastasis rates were significantly higher (37.93% vs 11.90% for liver metastasis, P=0.002; 44.83% vs 20.23 % for bone metastasis, P=0.010), and the incidence of thrombocytopenia was significantly increased (13.79% vs 1.19%, P=0.020) in patients with DTCs≥111 per 3 ml than those with DTCs＜111 per 3 ml. The positive rates of bone marrow liquid-based cytology in the disease control group and the disease progression group were 12.00% (12/100) and 46.15% (6/13), respectively, and the difference was statistically significant (P=0.002). However, the result of SE-iFISH revealed the DTCs quantities of the above two groups were 29 (8,110) and 64 (15,257) per 3 ml, and there was no statistical difference between the two groups (P=0.329). Univariate analysis depicted that the median progression-free survival (PFS) and median overall survival (OS) of liquid-based cytology positive patients were significantly shorter than those of tumor cell negative patients (6.33 months vs 9.27 months for PFS, P=0.019; 8.03 months vs 19.50 months for OS, P=0.019, P=0.033). The median PFS and median OS in patients with DTCs≥111 per 3 ml decreased significantly than those with DTCs＜111 per 3 ml (6.83 months vs 9.50 months for PFS, P=0.004; 11.2 months vs 20.60 months for OS, P=0.019). Multivariate analysis showed that disease stage (HR=2.806, 95%CI:1.499-5.251, P=0.001) and DTCs quantity detected by SE-iFISH (HR=1.841, 95%CI:1.095-3.095, P=0.021) were independent factors of PFS, while disease stage was the independent factor of OS (HR=2.538, 95%CI:1.169-5.512, P=0.019). Conclusions: Both bone marrow liquid-based cytology and SE-iFISH are clinically feasible. The positive detection of liquid-based cytology or DTCs≥111 per 3 ml was correlated with distant metastasis, and DTCs≥111 per 3 ml was an independent prognostic factor of decreased PFS in SCLC.

Prognostic value of disseminated tumor cells in unresectable pancreatic ductal adenocarcinoma: a prospective observational study

BackgroundAlthough pancreatic ductal adenocarcinoma (PDAC) rarely metastasizes to the skeleton, disseminated tumor cells have been detected in bone marrow samples from patients with this disease. The prognostic value of such findings is currently unclear. Thus, the current study aimed to clarify the prognostic information associated with disseminated tumor cell detection in samples from patients with PDAC.MethodsBone marrow aspirates were obtained from 48 patients with locally advanced (n = 11) or metastatic (n = 37) PDAC, before and after 2 months of chemotherapy. Disseminated tumor cells were detected with an mRNA panel and quantitative reverse transcription PCR. We used the highest levels measured in healthy bone marrow (n = 30) as a threshold to define the positive detection of disseminated tumor cells. Progression-free and overall survival were analyzed with Kaplan–Meier and Cox proportional hazards regression analyses.ResultsDisseminated tumor cells were detected in 15/48 (31%) bone marrow samples obtained before starting chemotherapy and in 8/25 (32%) samples obtained during chemotherapy. Patients with disseminated tumor cells detected before therapy had significantly shorter progression-free (p = 0.03; HR = 2.0) and overall survival (p = 0.03; HR = 2.0), compared to those without disseminated tumor cells in the bone marrow. When restricting disseminated tumor cell detection to keratins KRT7 and KRT8, the prognostic information was substantially stronger (p = 1 × 10–6; HR = 22, and p = 2 × 10–5; HR = 7.7, respectively). The multivariable Cox regression analysis demonstrated that disseminated tumor cell detection prior to treatment had independent prognostic value. In contrast, disseminated tumor cells detected during treatment did not have prognostic value.ConclusionsDisseminated tumor cells detected before commencing chemotherapy had prognostic value in patients with inoperable PDAC.

Neoadjuvant Chemotherapy of Patients with Early Breast Cancer Is Associated with Increased Detection of Disseminated Tumor Cells in the Bone Marrow.

Simple SummaryDisseminated tumor cells (DTCs) present in the bone marrow of breast cancer patients are an indicator of minimal residual disease and micrometastatic spread. These cells can already be found at the earliest disease stages and are associated with poorer outcomes. In preclinical models, neoadjuvant chemotherapy was shown to promote micrometastatic spread. The aim of this large single-center retrospective study was to compare the frequency and prognostic significance of DTC detection between patients treated with neoadjuvant chemotherapy and treatment-naive patients.Preclinical data suggest that neoadjuvant chemotherapy (NAT) may promote micrometastatic spread. We aimed to compare the detection rate and prognostic relevance of disseminated tumor cells (DTCs) from the bone marrow (BM) of patients with early-stage breast cancer (EBC) after NAT with that of therapy-naive EBC patients. DTCs were identified from BM samples, collected during primary surgery. Patients who received NAT were compared to patients who received chemotherapy after surgery. In total, 809 patients were analyzed. After NAT, 45.4% of patients were DTC-positive as compared to 19.9% of patients in the adjuvant chemotherapy group (p < 0.001). When sampled in patients who had undergone NAT, the detection of DTCs in the BM was significantly increased (OR: 3.1; 95% confidence interval (CI): 2.1–4.4; p < 0.001). After NAT, DTC-positive patients with ≥2 DTCs per 1.5 × 106 mononuclear cells in their BM had an impaired disease-free survival (HR: 4.8, 95% CI: 0.9–26.6; p = 0.050) and overall survival (HR: 4.2; 95% CI: 1.4–12.7; p = 0.005). The higher rate of DTC-positive patients after NAT as compared to a treatment-naive comparable control cohort suggests that NAT supports tumor cell dissemination into the bone marrow. DTC positivity in BM predicted relapse in various distant organs, implying that tumor cell dissemination was not restricted to the bone marrow.

Evaluation of the Hamburg-Glasgow Classification in Pancreatic Cancer: Preoperative Staging by Combining Disseminated Tumor Load and Systemic Inflammation.

Simple SummaryDespite the great achievements in pancreatic ductal adenocarcinoma (PDAC), identification of patients who will suffer rapid disease relapse and progression is not perfect, when definitive histology for tumor staging is not available. The Hamburg Glasgow Classification combines tumor cell dissemination in the bone marrow and systemic inflammatory response into a preoperative staging system. In this work, we assessed the Hamburg Glasgow Classification in potentially resectable PDAC as a prognostic classification for overall and progression-free survival and compared it to the UICC-TNM classification with promising results.This study aims to compare the Hamburg Glasgow Classification (HGC) to Union for International Cancer Control (UICC) classification in patients with pancreatic ductal adenocarcinoma (PDAC). As adequate tumor classification is only possible after tumor resection and histological evaluation, only 20% of patients with PDAC receive accurate tumor staging. Thus, an accurate preoperative staging system is still missing but urgently needed. Systemic inflammation and tumor dissemination are important factors regarding the oncological outcome. HGC integrates both into a preoperative staging system, by combining C-reactive protein (CRP), albumin, and disseminated tumor cells (DTC) in the bone marrow. In this prospective study, 109 patients underwent surgical exploration for suspected PDAC. All patients underwent a preoperative bone marrow aspiration for DTC detection. HGC showed significant preoperative risk stratification for overall survival (OS) (p-value < 0.001) and progression-free survival (PFS) (p-value < 0.001). These results were comparable to the UICC survival stratification for OS and PFS (p-value = 0.001 and 0.006). Additionally, in non-metastatic PDAC, HGC III-IV was associated with shorter OS and PFS (p-value < 0.001, respectively) when compared to HGC I-II. Therefore, the HGC is a promising preoperative prognostic staging classification for accurate and simple outcome stratification in patients with PDAC.

Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma.

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker‐positive cells were isolated by micromanipulation followed by single‐cell whole‐genome amplification and metaphase‐based comparative genomic hybridization (mCGH) to determine genome‐wide copy number alterations. The findings were correlated with clinical parameters and follow‐up data. We detected chromosomal aberrations in KRT18‐ and EpCAM‐positive cells from both compartments; BM‐derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow‐up. Genomic profiling of BM‐DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker‐positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM‐DTCs with high‐resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.

Disseminated tumour cells from the bone marrow of early breast cancer patients: Results from an international pooled analysis

PurposePresence of disseminated tumour cells (DTCs) in the bone marrow (BM) has been described as a surrogate of residual disease in patients with early breast cancer (EBC). PADDY (Pooled Analysis of DTC Detection in Early Breast Cancer) is a large international analysis of pooled data that aimed to assess the prognostic impact of DTCs in patients with EBC. Experimental designIndividual patient data were collected from 11 centres. Patients with EBC and available follow-up data in whom BM sampling was performed at the time of primary diagnosis before receiving any anticancer treatment were eligible. DTCs were identified by antibody staining against epithelial cytokeratins. Multivariate Cox regression was used to compare the survival of DTC-positive versus DTC-negative patients. ResultsIn total, 10,307 patients were included. Of these, 2814 (27.3%) were DTC-positive. DTC detection was associated with higher tumour grade, larger tumour size, nodal positivity, oestrogen receptor and progesterone receptor negativity, and HER2 positivity (all p < 0.001). Multivariate analyses showed that DTC detection was an independent prognostic marker for overall survival, disease-free survival and distant disease-free survival with hazard ratios (HR) and 95% confidence intervals (CI) of 1.23 (95% CI: 1.06–1.43, p = 0.006), 1.30 (95% CI: 1.12–1.52, p < 0.001) and 1.30 (95% CI: 1.08–1.56, p = 0.006), respectively. There was no association between locoregional relapse-free survival and DTC detection (HR 1.21; 95% CI 0.68–2.16; p = 0.512). ConclusionsDTCs in the BM represent an independent prognostic marker in patients with EBC. The heterogeneous metastasis-initiating potential of DTCs is consistent with the concept of cancer dormancy.

Analysis of disseminated tumor cells (DTCs) in primary breast cancer patients with various molecular subtypes.

e13000 Background: Despite successful treatment of the primary tumor, recurrence occurs in about 30% of breast cancer patients. One reason might be hematogenous spread during early disease stages. Disseminated breast cancer cells (DTCs) preferentially migrate into the bone marrow (BM) at early stages of the disease. Due to low proliferation, DTCs are persistent against systemic chemotherapy and might cause metastatic relapse at a later stage. Methods: BM aspirates were collected from the anterior iliac crest of patients with primary mamma carcinoma during surgery. After density gradient centrifugation cell suspensions were transferred onto glass slides and subjected to immunocytochemical staining against pan-cytokeratin. DTCs were visualized in pink using alkaline phosphatase and short counterstaining with hematoxylin which colored the nuclei light blue. DTCs were semi-automatically detected and enumerated using the Aperio Versa microscope based scanning system with a rare events algorithm that was trained to identify DTC candidates according to color, shape, intensity and size. As a positive control with each run, we used reference slides with a mix of bone marrow cells and a defined number of HCT116 cells. Results: Between February 2019 and December 2020 BM aspirates from 158 primary breast cancer patients were collected. Per patient about 4 million BM cells were analyzed. DTC detection revealed a positivity rate of 29% (46 patients). Molecular subtype analysis of DTC positive patients showed that 37% of the primary tumors (17 patients) were luminal A and 37% (17 patients) luminal B. In 9% of the cases (4 patients), tumors were HER2 enriched and 15% (8 patients) were triple negative. DTC count indicated that the majority of luminal B patients had 11-20 DTCs whereas luminal A patients tended to have lower DTC quantities varying between 1 and 10 DTCs. Conclusions: DTCs may serve as independent prognostic markers. Follow-Up data might reveal whether DTC quantification and molecular subtypes at primary diagnosis can be used to stratify patients at elevated risk for recurrence.

Abstract PS2-07: Outcomes associated with disseminated tumor cells at surgery after neoadjuvant chemotherapy in high-risk early stage breast cancer: The I-SPY SURMOUNT study

Abstract Background: Disseminated tumor cells (DTCs) in bone marrow detected after treatment may represent occult residual disease. We enumerated DTCs after neoadjuvant chemotherapy (NACT) in patients (pts) diagnosed with high-risk early stage breast cancer and examined the relationship of these cells with response and survival. Methods: I-SPY SURMOUNT is a sub-study of the I-SPY 2 TRIAL (NCT01042379). Pts enrolled on I-SPY 2, who signed consent for this sub-study, had bone marrow aspirates (BMA) collected after NACT at the time of surgery. DTCs were isolated and enumerated from BMA using immunomagnetic enrichment/flow cytometry (IE/FC). DTCs were defined as EPCAM-positive and CD45-negative nucleated cells. Samples were considered positive using a predetermined threshold of &amp;gt;4 DTCs per mL (Magbanua et al, unpublished data). Pathologic response was assessed using the residual cancer burden (RCB) method at local sites, and pts underwent standard adjuvant therapy if indicated and follow up for recurrence events and death. Relationship of DTCs with clinicopathologic variables was examined using Chi-squared test. Group means were compared using t tests. The log-rank test was used to compare survival curves. Results: A total of 73 patients were enrolled, 51 of whom had successful DTC assessment. The median DTC per mL was 4 (interquartile range 1.2-11.6). 24/51 (47%) were DTC-positive. Clinical characteristics by DTC status are shown in the table. DTC-positive pts were significantly younger (p=0.02) and had larger pretreatment tumors (longest diameter by magnetic resonance imaging) compared to DTC-negative pts (p=0.032). DTCs were not associated with receptor subtype. Thirty pts (41%) achieved a pathologic complete response (pCR). DTCs were not associated with pCR (p= 0.166); however, DTC-positive patients were significantly more likely to have residual cancer (RCB-II/III) after NACT compared to DTC-negative patients (OR 3.3, p=0.037). Median follow up of this cohort was 2.8 years (range: 0.9-4.8). Interim survival analysis showed that DTCs were not significantly correlated with EFS (p=0.6) or DRFS (p=0.41). Conclusions: Detection of DTCs at surgery after NACT is significantly more common in young patients, those with larger tumors, and those with residual disease at surgery. While these associations suggest higher risk for later recurrence, larger studies and longer follow up are necessary to determine if DTCs add prognostic value over pathologic evaluation alone for pts receiving NACT. Citation Format: Mark Jesus M Magbanua, Laura van 't Veer, Amy Clark, A. Jo Chien, Judy Boughey, Heather Han, Anne Wallace, Heather Beckwith, Minetta Liu, Christina Yau, E. Paul Wileyto, Lamorna Brown Swigart, Jane Perlmutter, Lauren Bayne, Shannon Deluca, Stephanie Yee, Erica Carpenter, Laura Esserman, John Park, Lewis Chodosh, Angela DeMichele. Outcomes associated with disseminated tumor cells at surgery after neoadjuvant chemotherapy in high-risk early stage breast cancer: The I-SPY SURMOUNT study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-07.

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.&#x0D; 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.&#x0D; 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.&#x0D; 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.&#x0D; 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+.

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.

Biochemical relapse in very high-risk prostate cancer after radical prostatectomy and DC-vaccine loaded with tumor RNA, hTERT, and survivin.

324 Background: Patients with very high-risk prostate cancer (VHR-PC) features experience worse outcome after radical prostatectomy. This study was designed to assess biochemical failure and toxicity of adjuvant dendritic cells vaccine (DCV) in prostate cancer patients who are at greatest risk for cancer progression. Methods: Twenty patients with pathological stage pT2 - pT3b and Gleason score 7B-10, pN0, pN+ or pNx were enrolled into the approved study DC-005. The primary end point was clinical failure. Ten patients were tested for disseminated tumor cells (DTCs) to the bone marrow before inclusion to the study. Three patients out of 10 patients had positive DTCs detection in bone marrow. The mean age of the cohort was 63 years (SD 6.9 years), and three patients had postsurgical pN1 status. Eighteen patients had two or more high-risk factors (ISUP grade 5, T3- stage and or PSA &gt; 20 ng/mL). Autologous dendritic cells were transfected with mRNA for hTERT, survivin and tumor mRNA. The DCV product was applied intradermally after curative intended surgery once per week the first months, then once per months the first year, thereafter every 3 months for two years or until biochemical progression (PSA relapse cut-off ≥ 0.3). Results: After 5 years follow-up (FU) 62% (12/20 patients) had not biochemically progressed and with a median FU of 69 months all patients included in the study are alive. Five patients were treated with salvage and one patient with adjuvant radiation treatment, three patients received limited ADT, and three patients are on first line ADT, none of those eight patients have experienced castration resistant prostate cancer. The toxicity was mild with no serious adverse event related to DCV. Conclusions: Adjuvant DCV mitigates the time to biochemical progression. These results appear favorably compared to historical controls in VHR-PC. The clinical outcomes of this study warrants a future enlarged clinical trial. Clinical trial information: NCT01197625.

Detection of disseminated tumor cells in bone marrow predict late recurrences in operable breast cancer patients

BackgroundOperable breast cancer patients may experience late recurrences because of reactivation of dormant tumor cells within the bone marrow (BM). Identification of patients who would benefit from extended therapy is therefore needed.MethodsBM samples obtained pre- and post-surgery were previously analysed for presence of disseminated tumor cells (DTC) by a multimarker mRNA quantitative reverse-transcription PCR assay. Updated survival analyses were performed on all patient data (n = 191) and in a subgroup of patients alive and recurrence-free after 5 years (n = 156). DTC data were compared to the mitotic activity index (MAI) of the primary tumors. Median follow-up time was 15.3 years.ResultsAmong the 191 patients, 49 (25.65%) experienced systemic relapse, 24 (49%) within 5–18 years after surgery. MAI and pre- and post-operative DTC status had significant prognostic value based on Kaplan–Meier analyses and multiple Cox regression in the overall patient cohort. With exclusion of patients who relapsed or died within 5 years from surgery, only pre-operative DTC detection was an independent prognostic marker of late recurrences. High MAI (≥10) did not predict late recurrences or disease-specific mortality.ConclusionPre-operative DTC detection, but not MAI status, predicts late recurrences in operable breast cancer.

Synchronous Detection of Circulating Tumor Cells in Blood and Disseminated Tumor Cells in Bone Marrow Predicts Adverse Outcome in Early Breast Cancer.

We examined the prognostic impact of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) detected at the time of surgery in 742 untreated patients with early breast cancer. DTCs in bone marrow were enumerated using the EPCAM-based immunomagnetic enrichment and flow cytometry (IE/FC) assay. CTCs in blood were enumerated either by IE/FC or CellSearch. Median follow-up was 7.1 years for distant recurrence-free survival (DRFS) and 9.1 years for breast cancer-specific survival (BCSS) and overall survival (OS). Cox regressions were used to estimate hazard ratios for DRFS, BCSS, and OS in all patients, as well as in hormone receptor-positive (HR-positive, 87%) and HR-negative (13%) subsets. In multivariate models, CTC positivity by IE/FC was significantly associated with reduced BCSS in both all (n = 288; P = 0.0138) and HR-positive patients (n = 249; P = 0.0454). CTC positivity by CellSearch was significantly associated with reduced DRFS in both all (n = 380; P = 0.0067) and HR-positive patients (n = 328; P = 0.0002). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n = 273), double positivity (CTC+/DTC+, 8%) was significantly associated with reduced DRFS (P = 0.0270), BCSS (P = 0.0205), and OS (P = 0.0168). In HR-positive patients, double positivity (9% of 235) was significantly associated with reduced DRFS (P = 0.0285), BCSS (P = 0.0357), and OS (P = 0.0092). Detection of CTCs in patients with HR-positive early breast cancer was an independent prognostic factor for DRFS (using CellSearch) and BCSS (using IE/FC). Simultaneous detection of DTCs provided additional prognostic power for outcome, including OS.

Abstract P3-01-02: Detection of circulating tumor cells (CTC) in blood and disseminated tumor cells (DTC) in bone marrow at surgery identifies breast cancer patients (pts) with long-term risk of distant recurrence and breast cancer-specific death

Abstract We examined the prognostic impact of CTCs and DTCs detected at the time of definitive surgery in pts diagnosed with early breast cancer (EBC). Methods: Blood and bone marrow samples from 742 treatment-naïve EBC pts, not eligible for neoadjuvant therapy, were collected immediately prior to surgery. 87% were hormone receptor (HR)-positive, and 71% were node-negative. DTCs (n=584) were enumerated using an EPCAM-based method involving immunomagnetic enrichment and flow cytometry (IE/FC). CTCs were enumerated either by IE/FC (n=288) or CellSearch (n=380). Optimal cutoffs for CTC-/DTC-positivity were selected using Monte-Carlo cross validation. Multivariate Cox regression analysis was performed to determine correlation between levels of CTCs/DTCs vs. distant recurrence-free survival (DRFS) and breast cancer-specific survival (BCSS). The overall median follow-up was 7.1 years for DRFS and and 9.1 years for BCSS, but extended up to 13.3 years in subset analyses (Table 1). Results: CTC-positivity by CellSearch was associated with HER2-positivity (Fisher p=0.01). Using optimized cutoffs in multivariate analyses, we found that CTC-positive pts by CellSearch had a statistically significant increased risk of distant recurrence (HR 4.93, p=0.0067). Moreover, pts who were CTC-positive by IE/FC had a statistically significant increased risk of breast cancer-specific death (HR=3.54, p=0.0138). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n=273), positive detection for both (CTC+DTC+) was significantly associated with increased risk of distant recurrence (HR=3.09, p=0.0270) and breast cancer-specific death (HR=4.55, p=0.0205). Table 1.Multivariate analysis to determine the prognostic significance of CTCs and DTCs detected at the time of surgery in treatment naive early breast cancer patients. Adjusted for age at diagnosis, tumor size, pathologic stage, HR and HER2 status, node status and grade. DRFS BCSS Variable and Method% positiveHR [95% CI]Wald p-valueMedian f/u [range] Years*HR [95% CI]Wald p-valueMedian f/u [range] Years*CTC+ vs. CTC- by CellSearch94.93[1.56-15.6]0.00676.4 [0.16-13.8]4.50[0.76-26.5]0.09627.5 [0.71-15.0]CTC+ vs. CTC- by IE/FC401.92[0.93-3.95]0.07599.8 [0.09-18.5]3.54[1.29-9.72]0.013813.3 [1.93-18.5]DTC+ vs. DTC- by IE/FC181.46[0.75-2.81]0.26317.5 [0.09-18.5]1.48[0.64-3.42]0.35429.8 [1.55-18.5]CTC+DTC+ vs. CTC-DTC- by IE/FC8**3.09[1.14-8.40]0.02709.8 [0.09-18.5]4.55[1.26-16.39]0.020513.3 [1.93-18.5]*f/u - follow-up; **double positive Conclusions: We demonstrate the impact of quantitative evaluation of CTCs and DTCs by IE/FC. Our large single institution dataset, in which CTCs and DTCs have been contemporaneously quantitated, has the longest patient follow-up. Simultaneous detection of CTCs and DTCs at the time of definitive surgery in treatment naïve EBC pts is an independent prognostic factor associated with increased long-term risk of distant recurrence and death due to breast cancer. Given the lack of early endpoints for low-risk patients, liquid biopsy may be an important consideration for future studies. Citation Format: Magbanua MJM, Yau C, Wolf D, Lee JS, Chattopadhyay A, Scott JH, Yoder E, Hwang S, Alvarado M, Ewing CA, Delson AL, van't Veer L, Esserman L, Park JW. Detection of circulating tumor cells (CTC) in blood and disseminated tumor cells (DTC) in bone marrow at surgery identifies breast cancer patients (pts) with long-term risk of distant recurrence and breast cancer-specific death [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-02.

Abstract GS5-07: International pooled analysis of the prognostic impact of disseminated tumor cells from the bone marrow in early breast cancer: Results from the PADDY study

Abstract Introduction As early breast cancer might relapse even after complete removal of breast and lymphnodes, the disease must persist in secondary sites. The detection of disseminated tumor cells (DTC) in the bone marrow (BM) has been described as a surrogate of residual disease. Various trials showed an impaired prognosis of DTC positive early breast cancer (EBC) patients. The PADDY (Pooled Analysis of DTC Detection in Early Breast Cancer) study is a large international pooled analysis that aimed to assess the prognostic impact of DTC detection in patients with EBC. Methods A pre-specified protocol was followed, and centers known to practice BM sampling for DTC detection were contacted for individual patient data. Patients with EBC, with available follow-up data and BM sampling before any anti-cancer treatment were eligible. BM aspirates were collected at the time of primary surgery. DTC were identified by antibody (A45-B/B3, AE1/AE3, 2E11 and E29) staining against cytokeratin. The DTC status was compared to other prognostic factors using the chi-squared test. Univariate log-rank test and multivariate cox regression were used to compare survival of DTC positive versus DTC negative patients. Results Individual data from 10,320 patients (11 centers from Europe and USA) were included with a median follow-up of 91 months. Of all patients, 2,823 (27.4 %) were DTC positive. DTC detection was associated with higher tumor grade, higher T stage, nodal positivity, ER and PR negativity, and HER2 positivity (all p&amp;lt;0.001). In univariate analyses, overall, breast cancer specific, disease-free and distant disease-free survival (OS, BCSS, DFS, DDFS) were significantly shorter in DTC positive patients with p-values of &amp;lt;0.001. Multivariate analyses showed the DTC status to be an independent prognostic marker for OS, BCSS, DFS and DDFS with hazard ratios (HR) and 95%-confidence intervals (CI) of 1.23 (95%-CI: 1.06-1.42, p=0.007), 1.38 (95%-CI: 1.11-1.72, p=0.004), 1.29 (95%-CI: 1.10-1.50, p=0.001) and 1.32 (95%-CI: 1.10-1.58, p=0.003), respectively. Conclusions Detection of DTC in the bone marrow is an independent prognostic marker in patients with non-metastatic breast cancer. Further studies should investigate the impact of DTC on metastatic cancer progression and their role for clinical decision making. Citation Format: Hartkopf AD, Brucker SY, Taran F-A, Harbeck N, von Au A, Naume B, Pierga J-Y, Hoffmann O, Beckmann MW, Rydén L, Fehm T, Aft R, Montserrat S, Walter V, Rack B, Schuetz F, Borgen E, Ta M-H, Bittner A-K, Fasching P, Fernö M, Krawczyk N, Weilbaecher K, Margelí M, Hahn M, Jueckstock J, Domschke C, Bidard F-C, Kasimir-Bauer S, Schoenfisch B, Kurt AG, Wallwiener M, Gebauer G, Wallwiener D, Janni W, Pantel K. International pooled analysis of the prognostic impact of disseminated tumor cells from the bone marrow in early breast cancer: Results from the PADDY study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS5-07.

DNA-Oriented Shaping of Cell Features for the Detection of Rare Disseminated Tumor Cells.

Metastasis of tumors is the major cause of death in cancer patients. The efficient detection of disseminated tumor cells (DTCs) is found to be critical for the early warning of tumor metastasis. However, it is technically difficult to identify DTC among circulation tumor cells (CTCs). In this work, we have proposed a DNA-oriented shaping strategy to convert the metastasis-associated feature of CTC into a dominant signature, making DTCs discernible. In detail, by performing the in situ DNA rolling circle amplification, invasive biomarkers at cell surface are specifically labeled with large amount of biotin-incorporated DNA strands. Therefore, isolation and detection processes of DTCs are significantly simplified. On a streptavidin immobilized ITO electrode, we demonstrated that these biotin-featured DTCs are efficiently captured, and as few as two cells can be electrochemically detected, with a linear detection range from 5 to 320 cells/cm2. Therefore, on the basis of the DNA-oriented shaping of cell, detection of DTCs becomes simple and sensitive, which may facilitate the process of rare DTC analysis. Besides, it is also promising to distinguish other cell types with this method in fields such as cell-based clinical diagnosis, systematic cell census, and tissue engineering.

DIsseminated tumor cells of luminal breast cancer patients .

Introduction. There is ample evidence that disseminated tumor cells (DTC), which are found in the bone marrow (BM) of patients with breast cancer (BC), including early stages, are progenitors of subsequent distant metastasis. Therefore, BM-DTC represent an additional tool for understanding carcinogenesis and estimating prognosis. Nevertheless, the existing data are controversial. The purpose of the study – to determine the frequency of DTC detection in BM of patients with luminal BC and also its relationship with some clinical and immunophenotypic parameters. Materials and methods. BM bioptates of 65 luminal BC patients were analyzed for the presence of DTC by Attune Acoustic Focusing Cytometer. For the first time in Russia, the sensitivity of the DTC detection method in the BM to the level of 1 × 10–7 myelocaryocytes was increased. Results. In BM, DTC were detected in 40 % of patients, and this finding did not correlate with stage of BC and degree of malignancy. The level of CD8+ lymphocytes in patients with DTC in the BM was significantly lower and amounted to 39,2 % versus 48,1 % in patients without DTC (p = 0,011). The content of myelocaryocytes with DTC-positive status was 1,6 times lower than in the absence of DTC (р = 0,007). Other parameters of the myelogram did not differ significantly. Moreover, no significant correlations were found between the presence of DTC in BM and the breast tumor immunophenotype (HLA-I: p = 0,74; HLA-DR: p = 0,93; CD71: p = 0,46). Conclusion. The presence of BM-DTC is more interrelated with myelogram and subpopulation of BM lymphocytes than with the clinical characteristics of tumor.

Disseminated Tumor Cells Predict Efficacy of Regional Nodal Irradiation in Early Stage Breast Cancer

Disseminated tumor cells (DTCs) collect in the bone marrow and indicate micrometastatic spread. We previously reported that DTCs could be a predictive factor for the efficacy of regional node irradiation (internal mammary nodes [IMNs]/supra- and infraclavicular nodes [SCNs]). In this article, we report the long-term results (>10years) on the impact of DTC status in early stage breast cancer. Patients with localized breast cancer were eligible for inclusion in this prospective cohort. DTCs were obtained from a medullary iliac crest sample performed before any primary therapy. DTC status was prospectively assessed by pathologists. Irradiation volumes were defined per standard of care. Cumulative incidence rates and hazard ratios were obtained using both Cox and Fine-Gray models. Interaction tests were performed to confirm the predictive value of DTC status in a multivariate analysis. Six hundred twenty patients with localized breast cancer were included. Overall, 94 patients (15.2%) were DTC-positive. After a median follow-up of 11.7years, 47 patients (7.6%) experienced locoregional relapse. DTC detection was associated with a higher risk of locoregional relapse in univariate and multivariate analyses (Cox hazard ratio, 3.26; 95% confidence interval, 1.6-5.7; P=.001). In the multivariate subgroup analysis, IMN/SCN irradiation significantly reduced locoregional relapse among DTC-positive patients compared with DTC-negative patients (interaction test: hazard ratio, 0.3; 95% confidence interval, 0.1-0.9; P=.02). IMN/SCN was the only irradiation volume with an impact on locoregional relapse in patients according to DTC status, and the predictive value of DTC status for the benefit of locoregional irradiation was independent of locoregional nodal status. This long-term analysis confirms the predictive impact of DTC status on the efficacy of regional radiation therapy for locoregional relapse in early breast cancer. After further studies, DTC status could be used as a decision tool to better tailor adjuvant radiation therapy in patients with early stage breast cancer.

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.

Abstract 5186: Overexpression of CXCR4 and JUNB in disseminated tumor cells (DTCs) isolated from breast cancer patients before neoadjuvant treatment

Abstract Introduction: The presence of Disseminated (DTCs) and Circulating Tumor Cells (CTCs) in the bone marrow (BM) and the blood of breast cancer (BC) patients are poor prognostic factors. We have recently shown that CTCs derived from metastatic BC patients, overexpressed CXCR4 and JUNB and this expression was related to clinical outcome. In this study we applied the same methodology to BM of non-metastatic BC patients at primary diagnosis, before the onset of therapy. Methods: Bilateral BM (10 ml) aspirations were assessed for the detection of DTCs by a Ficoll density gradient centrifugation and subsequent centrifugation of the BM cells onto glass slides [1x106 mononuclear cells (MNCs) per slide]. Triple staining experiments with panytokeratin/CXCR4/JUNB antibodies were performed. An expression pattern of the examined proteins was created, using three different BC cell lines (SKBR3, MDA-MB231, and MCF7). Consequently, 10 BM aspirations from BC patients were analyzed. Mean intensity for each examined molecule was calculated automatically with the ARIOL system. Results: Quantification of CXCR4 among BC cell lines revealed that CXCR4 expression followed the subsequent hierarchy SKBR3&amp;gt;MCF7&amp;gt;MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231 cells, respectively. Accordingly, the expression pattern of JUNB in cell lines was SKBR3&amp;gt;MCF7&amp;gt;MDA-MB231. Consequently, we analyzed 10 BM samples from primary BC patients. The (CXCR4+JUNB+CK+) phenotype was observed in 90% (9/10) of the samples. In addition, the (CXCR4-JUNB+CK+) phenotype was detected in 20% (2/10) of the patients. Interestingly, the (CXCR4+JUNB-CK+) phenotype was not observed in any of the patient's samples. Quantification of CXCR4 expression revealed that the mean intensity in DTCs (19.1±2) was statistically higher than in MCF7 (13±0.51; p=0.006) and MDA-MB231 (10.22±0.7; p= 0.0002) cell lines. It was also higher compared to normal donors' PBMCs (10.6±1.01; p=0.0004). In addition, the mean intensity of JUNB was statistically increased in DTCs (17.1±1.66) compared to MCF7 (8.9±0.56; p=0.00001) and MDA-MB231 (6.6±0.15; p= 0.00001) cells, respectively. Furthermore, it was increased compared to normal donors' PBMCs (5.2±0.15; p=0.0002). In two patients, we had available samples after treatment. In one patient, DTCs were eliminated after neoadjuvant therapy, for the other patient, DTCs were reduced from 10 to two DTCs. However, these cells were double positive for CXCR4 and JUNB. Conclusion: CXCR4 and JUNB were overexpressed in DTCs derived from primary BC patients, in agreement to previous findings in CTCs, implying an upregulation of the CXCR4 pathway in these cells. Quantification of this expression potentially defines a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from targeted therapies. Citation Format: Galatea Kallergi, Oliver Hoffmann, Nefeli Zacharopoulou, Christos Stournaras, Vassilis Georgoulias, Sabine Kasmir-Bauer. Overexpression of CXCR4 and JUNB in disseminated tumor cells (DTCs) isolated from breast cancer patients before neoadjuvant treatment [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 5186.