Disseminated Tumor Cells In Bone Marrow Research Articles

Abstract P5-08-01: Pilot study of a patient-reported outcome (PRO) measurement strategy to determine impact of screening for minimal residual disease (MRD) in high-risk breast cancer survivors

Abstract Background: Patients treated for early stage breast cancer (BC) have a 30% lifetime risk of developing metastatic disease. Numerous studies have demonstrated that dormant bone marrow disseminated tumor cells (DTCs) are independently associated with risk of recurrence and death, yet interventions targeting these cells are lacking. The PENN-SURMOUNT (Surveillance Markers of Utility for Recurrence after (Neo)adjuvant Therapy) Screening Study was launched in 2016 to screen high risk BC survivors for DTCs using bone marrow aspirate (BMA) and identify eligible DTC positive patients for clinical trials. Given the novelty of this approach, we concurrently developed and pilot tested a PRO measurement strategy to study how the screening method of BMA and disclosure of DTC results impacts early-stage BC patients. Methods: PENN-SURMOUNT is a single center prospective, longitudinal cohort study examining BM and blood biomarkers of MRD among patients within 5 years of BC diagnosis who have high risk criteria (positive axillary nodes, triple negative biology, ER+ with Oncotype Dx ≥ 25 and/or high risk Mammaprint, or pathologic residual disease after neoadjuvant chemotherapy). From May 2019 – August 2021, we recruited patients on SURMOUNT to complete PRO surveys at baseline (T0), after BMA (T1), and after disclosure of DTC results (T2). Surveys were administered in paper form initially, then electronic form starting Feb 2021. PRO survey instruments were selected through literature review, followed by consensus among multidisciplinary clinical and research experts and patient advocates. PRO measures assess recurrence distress (Quality of Life in Adult Cancer Survivors, QLACS), illness intrusiveness (Illness Intrusiveness Ratings Scale, IIRS), and decision making (Decision Regret Scale). Additional survey items assess tolerability of the BMA and patients’ risk perception and cognitive understanding after DTC results disclosure. Descriptive statistics summarize PRO survey compliance and responses at T0, T1, and T2 in the total population and the population who reported longitudinal data for T2. Results: 61 of 66 eligible patients on the SURMOUNT trial enrolled in the PRO pilot study and completed a baseline survey, of which 47 (77%) tested negative for DTCs. Mean completion rates were 0.92 at T0, 0.85 at T1, and 0.56 at T2. After electronic survey implementation, completion rates increased to 0.94 (T0), 0.97 (T1) and 0.81 (T2). At T0, 36 (59%) patients reported a high risk perception of developing BC recurrence at 5 years and 42 (69%) during their lifetime. Mean T0 recurrence distress using the QLACS subscale was 14.6 (SD 6.3) out of possible score 4-28, compared to an expected mean of 11.42 (SD 5.48) in a general survivorship population. Mean T0 illness intrusiveness was 27.3 (SD 13.9) out of possible score 13-91. At T1, approximately 85% of patients agreed that they correctly understood the purpose of the bone marrow procedure and what the procedure would entail. 44 (72%) of patients reported a maximum pain score <= 4 in the week post-procedure and 42 (69%) reported the BMA was same or better than expected tolerability. Exploratory subset analysis of patients with complete longitudinal data at T2 (n = 34) showed average scores of 13.4 (SD 6.0), 30.1 (SD 14.0), and 2.8 (SD 6.2) for recurrence distress, illness intrusive, and decision regret scores (scale 0-100), respectively. At T2, 26 (76%) of patients reported no decision regret for undergoing testing for DTCs; 27 (79%) reported feeling less anxious after DTC results disclosure. Conclusions: Participants of PENN-SURMOUNT perceived risk of recurrence as high. The BMA procedure was well-tolerated and better than expected among the majority of this cohort, and most did not regret having undergone BMA after DTC status disclosure. Longitudinal completion rates were low, limiting assessment of PROs at later time points, a major focus of future work in this setting. Citation Format: Tara Kaufmann, Patrick Chang, Shoshana Rosenberg, Elizabeth Frank, Brian Hobbs, Lauren J. Bayne, Isoris Nivar, Brooke L. Goodspeed, Killian M. Rohn, Emily M. Kugler, Kevin Fox, Susan Domchek, Angela Bradbury, Payal Shah, Hayley Knollman, Rachel C. Jankowitz, Igor Makhlin, Amy S. Clark, Lewis A. Chodosh, Angela DeMichele, Katherine Goodfellow. Pilot study of a patient-reported outcome (PRO) measurement strategy to determine impact of screening for minimal residual disease (MRD) in high-risk breast cancer survivors [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 P5-08-01.

A microfluidic-based filtration system to enrich for bone marrow disseminated tumor cells from breast cancer patients.

Disseminated tumors cells (DTCs) present in the bone marrow (BM) are believed to be the progenitors of distant metastatic spread, a major cause of mortality in breast cancer patients. To better understand the behavior and therapeutic vulnerabilities of these rare cell populations, unbiased methods for selective cell enrichment are required. In this study, we have evaluated a microfluidic-based filtration system (ParsortixR, Angle PLC), previously demonstrated for use in circulating tumor cell (CTC) capture, to capture BM DTCs. Performance using BM samples was also compared directly to enrichment of CTCs in the peripheral blood (PB) from both metastatic and non-metastatic breast cancer patients. Although the non-specific capture of BM immune cells was significant, the device could routinely achieve significant cytoreduction of BM and PB WBCs and at least 1,000-fold enrichment of DTCs, based on labeled tumor cell spike-in experiments. Detection of previously characterized DTC-associated gene expression biomarkers was greatly enhanced by the enrichment method, as demonstrated by droplet digital PCR assay. Cells eluted from the device were viable and suitable for single cell RNA sequencing experiments. DTCs in enriched BM samples comprised up to 5% of the total cell population, allowing for effective single cell and population-based transcriptional profiling of these rare cells. Use of the Parsortix instrument will be an effective approach to enrich for rare BM DTCs in order to better understand their diverse molecular phenotypes and develop approaches to eradicate these cells to prevent distant disease development in breast cancer 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+.

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.

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.

Abstract P2-12-01: Patient attitudes, experience and results of screening for minimal residual disease (MRD) for therapeutic intervention

Abstract Background: Patients (pts) treated for early stage breast cancer (BC) have a 30% lifetime risk of developing incurable, distant metastatic disease. Yet, standard monitoring after definitive therapy for primary disease is passive observation. Numerous studies have demonstrated that dormant bone marrow (BM) disseminated tumor cells (DTCs) are independently associated with recurrence, but assessment of DTCs is not performed in clinical practice, largely because of concerns about the acceptability and logistics of bone marrow aspiration (BMA) and lack of established therapies that target DTCs. As part of a large scale screening study for a clinical trial targeting DTCs, we examined pt attitudes about DTC screening and subsequently assessed feasibility and tolerability of BM DTC assessment. Methods: The PENN-SURMOUNT (Surveillance Markers of Utility for Recurrence after (Neo)adjuvant Therapy) Screening Study is a single center prospective, longitudinal cohort study examining BM and blood biomarkers of minimal residual disease (MRD) among pts within 5 years of BC diagnosis, who meet one of the following high risk criteria: positive axillary nodes, triple negative biology, ER+ with Oncotype Dx ≥ 25 and/or high risk Mammaprint, or residual disease (RD) after neoadjuvant chemotherapy (NACT). Consented pts undergo a baseline outpatient BMA; if negative, pts can repeat screening annually. During trial design, we surveyed 25 women with stage 2-3 BC at random from the breast clinic at the University of Pennsylvania to assess feasibility. On the SURMOUNT Study, we collected demographic and clinical characteristics of pts, and patient-centered survey data regarding feasibility and acceptability of the BMA that is administered within 48 hours of the procedure. Results: In the pre-trial feasibility survey, 21/25 (84%) pts indicated they were very/definitely interested in knowing if they harbored DTCs. Of those, 18 (86%) indicated moderate/definite interest in testing for DTCs with BMA after the BMA was described to them in detail. 20 (95%) of pts indicated moderate/definite interest in taking oral therapy to eradicate DTCs. 14 (67%) pts stated undergoing up to 3 additional BMA would not change their likelihood of undergoing the clinical trial; only 1 stated much less likely. In the subsequent SURMOUNT study, 361 pts have been referred to date; 167 were eligible, and 136 (81%) subsequently enrolled. 21 (13%) are still in screening. 130 pts have had at least 1 BMA with annual re-screens in 37 (year 1) and 8 (year 2). 39% traveled >50 miles to participate. Post-BMA symptoms were rare (bleeding 2%; redness 12%) though 59%/70% reported mild-moderate pain/tenderness. After BMA, 47%/29%/25% reported it was better/same/worse than expected. 30%/32%/22% reported minimal/moderate/high anxiety prior to the BMA. Afterward, only 20%/5%/4% reported minimal/moderate/high residual anxiety. In 128 pts with results, 38 (30%) have ≥ 1 DTC (30 initial, 8 on follow-up); by risk group: 20/77 (26%) node positive, 20/64 (31%) triple negative, 3/8 (38%) ER+/RS≥25, 7/29 (24%) with RD post-NACT. DTC+ pts were similar to DTC- in median age (50.4), race, distance traveled, menopausal status and BMI. 94% of DTC+ pts have entered the CLEVER clinical trial. Conclusion: A majority of BC survivors want to know DTC status; a majority of these are willing to have BMA and enroll on a clinical therapeutic trial, many are willing to travel to participate and are willing to undergo annual BMA assessment. The SURMOUNT study shows that screening for DTCs is feasible and effective in identifying pts for therapeutic intervention targeting MRD to reduce recurrence. Citation Format: Isoris Nivar, Tara Kauffman, Lauren Bayne, Paul Wiley, Brooke Goodspeed, Michael Feldman, Lewis Chodosh, Amy Clark, Angela DeMichele. Patient attitudes, experience and results of screening for minimal residual disease (MRD) for therapeutic intervention [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-12-01.

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.

Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer.

Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.

Bone Marrow Involvement in Melanoma. Potentials for Detection of Disseminated Tumor Cells and Characterization of Their Subsets by Flow Cytometry.

Disseminated tumor cells (DTCs) are studied as a prognostic factor in many non-hematopoietic tumors. Melanoma is one of the most aggressive tumors. Forty percent of melanoma patients develop distant metastases at five or more years after curative surgery, and frequent manifestations of melanoma without an identified primary lesion may reflect the tendency of melanoma cells to spread from indolent sites such as bone marrow (BM). The purpose of this work was to evaluate the possibility of detecting melanoma DTCs in BM based on the expression of a cytoplasmatic premelanocytic glycoprotein HMB-45 using flow cytometry, to estimate the influence of DTCs’ persistence in BM on hematopoiesis, to identify the frequency of BM involvement in patients with melanoma, and to analyze DTC subset composition in melanoma. DTCs are found in 57.4% of skin melanoma cases and in as many as 28.6% of stage I cases, which confirms the aggressive course even of localized disease. Significant differences in the groups with the presence of disseminated tumor cells (DTCs+) and the lack thereof (DTC−) are noted for blast cells, the total content of granulocyte cells, and oxyphilic normoblasts of erythroid raw cells.

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.

Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells

BackgroundCancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal.ResultsWe have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow.ConclusionsRobust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.

Abstract 55: Intrinsic molecular characteristics of bone marrow-disseminated tumor cells

Abstract Dormant or slow-cycling disseminated tumor cells (DTC) in bone marrow (BM) are known to persist in patients with various types of cancer including head and neck squamous cell carcinoma (HNSCC). BM-DTC has been thought to have possible metastatic stem cell traits although functional and clonal analysis of BM-DTC remain elusive. Recently, we reported that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis was crucial for drug resistance dependent on a slow-cycling state in BM-DTC but not in lung metastatic cells. This study aimed to elucidate intrinsic molecular characteristics in BM-DTC. We used the human HNSCC cell line HEp3-originated sublines [i.e. parental line (P-HEp3), BM-DTC-derived (BM-HEp3), and lung metastases-derived sublines (Lu-HEp3)]. Our transcriptome analyses for these sublines revealed that slow-cycling and drug-resistant BM-HEp3 cells had unique gene expression signatures. In addition, our investigation for phosphorylation of 39 kinases associated with tumor progression showed enhanced activation of only Src in BM-HEp3 compared with P- and Lu-HEp3. Importantly, Src inhibition by dasatinib in these sublines led to a remarkable reduction in cell viability only in BM-HEp3. These data suggest that intrinsic molecular characteristics of BM-DTC are distinct from primary and lung metastatic tumor cells. Src activation may be important for survival of dormant or slow-cycling BM-DTC. Further studies should contribute to better understanding of biology of minimal residual disease in BM and metastatic initiation. Citation Format: Manabu Maeshiro, Satoru Shinriki, Takuya Nakamura, Hirofumi Jono, Hideki Nakayama, Yukio Ando, Hirotaka Matsui. Intrinsic molecular characteristics of bone marrow-disseminated tumor cells [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 55.

Parallels between hematopoietic stem cell and prostate cancer disseminated tumor cell regulation.

The bone marrow is the primary site of hematopoiesis and the home for hematopoietic stem cells (HSCs) in adult mammals. Prostate cancer commonly metastasizes to the bone and forms bone metastases in almost all patients who die of the disease. Prostate cancer bone metastases are thought to develop after rare bone marrow disseminated tumor cells (DTCs) escape a dormant state and reactivate. Prostate cancer DTCs and normal HSCs have been shown to compete for residence in the bone marrow and share many of same regulatory mechanisms for survival, proliferation and homing. In this review, we highlight these parallels in order to help our readers use the literature in HSC and DTC biology to inform their research and generate hypotheses in both fields.

Tracing the Seeds in the Soil.

Featured Article: Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC et al. A pooled analysis of BM micrometastasis in breast cancer. N Engl J Med. 2005;353:793–802.2 In our article featured here, we reported in a pooled analysis of >4700 patients with primary breast cancer (stage M0) that the presence of disseminated tumor cells (DTCs)3 in the bone marrow (BM) at the time of diagnosis was an independent predictor of poor prognosis. Metastasis formation in secondary organs, which is the major cause of death from solid tumors, originates from tumor seeds released from the primary lesion before it is removed by the surgeon. Our initial efforts to trace DTCs in BM were largely motivated by the fact that the bones are major sites of distant metastasis in breast cancer (and other solid tumors such as prostate and lung cancer). Moreover, the BM is accessible by needle aspiration, an invasive but safe diagnostic approach used for decades in patients with hematological malignancies. Finally, the BM is a mesenchymal organ, and breast cancers (as most other …

A Facile, In Vitro 384-Well Plate System to Model Disseminated Tumor Cells in the Bone Marrow Microenvironment.

Bone marrow disseminated tumor cells (DTCs) are dormant cancer cells that harbor themselves in a bone marrow niche for years after patient remission before potentially returning to a proliferative state, causing recurrent cancer. DTCs reside in bone marrow environments with physiologically important mesenchymal stem cells that are often negatively affected by chemotherapy treatments. Currently, there are very few models of DTCs that recapitulate their dormant phenotype while producing enough samples to accurately quantify cancer and surrounding stromal cell behaviors. We present a three-dimensional spheroid-based model system that uses dual-color bioluminescence imaging to quantify differential cell viability in response to various compounds. We successfully screened for compounds that selectively eliminated cancer cells versus supportive stromal cells and verified results with comparison to efficacy in vivo. The spheroid coculture system successfully modeled key aspects of DTCs in the bone marrow microenvironment, facilitating testing for compounds to selectively eliminate DTCs.

Long-Term Impact of Bone Marrow Disseminated Tumor Cells Detection of Breast Cancer Locoregional Relapses

Long-Term Impact of Bone Marrow Disseminated Tumor Cells Detection of Breast Cancer Locoregional Relapses

Abstract 4942: Small molecule inhibition of CXCR4 mobilizes prostate disseminated tumor cells (DTCs) from the chemoprotective bone marrow niche, thereby sensitizing them to chemotherapy

Abstract Bone metastasis results in the deaths of approximately 28,000 prostate cancer patients each year in the United States. Disseminated tumor cells (DTCs) go from the primary tumor site to the bone marrow, which provides a chemoprotective environment. C-X-C chemokine receptor 4 (CXCR4), highly overexpressed in prostate cancer, is at least partially responsible for bone homing. CXCR4 is also expressed in hematopoietic stem cells (HSCs), which occupy the same niche within the bone marrow as DTCs. HSCs can be mobilized from the bone marrow niche via CXCR4 antagonism. We have previously shown that human tumor cells can be similarly mobilized from the bone marrow niche via small molecule inhibition of CXCR4. We believe this represents an ideal strategy for treating bone metastasis and hypothesize that mobilized DTCs will proliferate in the vascular niche, causing them to become chemosensitive. Several outstanding questions still exist that we are in the process of answering. First is the percentage of DTCs that are mobilized with CXCR4 antagonism. Previously, we used a qPCR strategy to quantify tumor cells in the bone marrow and blood of mice. This was imperfect because we could not reliably count cells, nor could we query the cells further. Therefore, we developed a novel immunofluorescence protocol to detect, quantify, and query tumor cells residing in the bone marrow or blood of mice. We will use this technique to determine the percentage of bone marrow DTCs that are mobilized. The second outstanding question is whether or not mobilized DTCs proliferate once mobilized. This is crucial to know, considering most chemotherapy relies on tumor cell proliferation. Using our novel protocol, we will determine the percentage of bone marrow DTCs and mobilized DTCs that are proliferating. Finally, while there is evidence that combining CXCR4 antagonism with chemotherapy kills more tumor cells than chemotherapy alone, no one has shown definitively that it works specifically for prostate bone marrow DTCs or bone metastasis. Not only will we do this, but we will also be able to quantify how effective each therapy strategy is by quantifying the tumor cells present in each scenario. The data from these in vivo murine experiments, which would be impossible to get from humans, will go hand in hand with a first-in-prostate cancer clinical trial we are running that tests the same hypothesis. Combined, these data will be critically important in deciding whether mobilization therapy combined with chemotherapy is an effective strategy for treating metastatic prostate cancer. Citation Format: Kenneth C. Valkenburg, Emma E. van der Toom, Kenneth J. Pienta. Small molecule inhibition of CXCR4 mobilizes prostate disseminated tumor cells (DTCs) from the chemoprotective bone marrow niche, thereby sensitizing them to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2017-4942

Abstract 4945: Axl-hi DTCs in the bone marrow of a genetically engineered mouse model of prostate cancer exhibit decreased proliferation

Abstract The majority of prostate cancer (PCa) deaths are attributed to bone metastases, which may not arise until years after the primary tumor has been removed. Disseminated tumor cells (DTCs) have been shown to already be present in the bone marrow of patients at the time of prostatectomy, and it is thought that they undergo a period of dormancy, allowing them to remain undetected for years before manifesting into clinically detectable metastases. It is not known what allows PCa DTCs to undergo and persist in this dormant state, but it is likely that factors in the bone marrow niche play an essential role. Previously our group has shown that one of these niche factors, Gas6, can regulate growth of PCa cells. The growth restraining effects of Gas6 on PCa cells is likely mediated through the tyrosine kinase receptor Axl, whose expression was found to be increased on dormant DTCs compared to proliferating metastases in a xenograft mouse model. Here, we utilized the TRAMP model, a genetically engineered mouse model of spontaneous PCa progression, to compare the frequency of Axl-hi and Axl-low bone marrow DTCs that are proliferating versus non-proliferating. Further, we also compared Axl expression and proliferation in matched primary tumors. To investigate the functional significance of Gas6/Axl signaling in mediating PCa dormancy, we have generated Axl knockout PCa cells to test the requirement of Axl in maintaining dormancy, and Axl overexpressing PCa cells to test the sufficiency of Axl in mediating dormancy. Determining the role of Axl and any other regulatory mechanisms of PCa DTC dormancy will be crucial in efforts to prevent lethal and incurable metastasis. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Brian W. Simons, Kenneth J. Pienta. Axl-hi DTCs in the bone marrow of a genetically engineered mouse model of prostate cancer exhibit decreased proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2017-4945

Long term follow up (LTFU) of adjuvant zoledronic acid (ZOL) in high risk early stage breast cancer (ESBC) defined by bone marrow (BM) disseminated tumor cells (DTC).

e12001 Background: BM DTC & peripheral blood CTC indicate metastatic potential. Adjuvant bisphosphonates in postmenopausal patients (pts) result in less bone metastases and better survival. We studied ZOL’s impact on DTC, CTC, and outcomes in ESBC (ASCO, 2010) and now report updated LTFU. Methods: Forty-five pts with ESBC and DTC > 4/mL BM were given ZOL every 4 weeks for 24 months (mo). DTC were quantified at baseline (BL), 12 and 24 mo of ZOL with immunomagnetic enrichment and flow cytometry (Clin Cancer Res, 2015). Serial CTC, survival, recurrence and toxicity were studied. Results: Forty-five pts received ZOL (56% HR+, 18% HER2+, 40% TN, 96% prior chemo). BL median DTC was 13.3/mL (range 4-333). Median DTC decreased from BL to 12 mo (median reduction 6.5/mL, range -57.2 to 23.6) and from BL to 24 mo (median reduction 4.6/mL, range -43.1 to 160.1). For 33 pts with BM at all 3 times, DTC decreased: BL median 11.5/mL (range 4-59.3), 12 mo median 6.8/mL (range 0-28.5), 24 mo median 6.5/mL (range 0.3-168.6). At 12 & 24 mo, 32% & 26% had undetectable DTC respectively. Median CTC was 0.2/mL at BL (n = 45), 0.25/mL at 6 mo (n = 30), 0/mL at 12 mo (n = 22) & 0.2/mL at 24 mo (n = 23). At 123 mo median FU (range 5-154), 9 (20%) pts recurred. Median time to recurrence was 21 mo (range 6-105); 67 % (6/9) TN, 22% (2/9) ER+/HER2+. The first sites of recurrence were CNS in 33% (3), bone & viscera in 22% (2), viscera in 22% (2) and local in 22% (2). BL DTC > 30/mL was associated with higher recurrence (50% > 30 vs. 14% < 30, p = 0.039). All 3 pts with CNS disease had BL DTC > 30/mL. Of the pts who recurred, 4 had ≥ 1 serial BMA; 2/4 had increasing DTC before recurrence. Of 32 disease free pts with BMA at all 3 times, 72% (23) had decreased DTC from BL to 24 mo. Of the 13.3% (6) pts who died, 67% (4) had BL DTC > 30/mL, which was associated with a greater risk of death (50% > 30 vs. 5% < 30, p = 0.006). BL CTC > 0.8/mL was associated with higher recurrence (60% > 0.8 vs. 15% < 0.8, p = 0.047) & death (60% > 0.8 vs. 8% < 0.8, p = 0.013). ZOL was well tolerated. Conclusions: At 123 mo FU, BL DTC > 30/mL and CTC > 0.8/mL correlated with recurrence & death. In this single arm phase II study, adjuvant ZOL decreased DTC over time, supporting the anti-tumor effect of bisphosphonates. Clinical trial information: NCT00295867.

Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration

PurposeMolecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.MethodsBM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.ResultsFiltering an average of 14×106 nucleated BM cells yielded approximately 17–21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84–92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.ConclusionsWe have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.