Martin, Stuart S.

Abstract Background: Circulating cancer associated macrophage-like cells (CAMLs) are cancer specific giant cells circulating in the blood of patients with solid tumors. Since their discovery, few studies have been done to elucidate their lineage or phenotypic identity. The difficulty in classifying CAMLs is exemplified by their expression of multiple heterogeneous markers that defy conventional identification. Recently, we described a restaining method (QUAS-R) to screen individual cells using an array of up to 15 biomarkers. We used this method to screen CAMLs isolated from 152 cancer patient samples in 4 types of solid tumors to classify CAMLs by phenotypic immunostaining. These data suggest that CAMLs are a morphologically diverse and phenotypically heterogeneous population of cancer specific giant cells with overlapping myeloid, epithelial, and endothelial phenotypes. Methods: This multi-institutional study used peripheral blood samples from 152 cancer patients (stage I-IV) from breast (n=42), lung (n=39), renal cell carcinoma (36) and prostate (n=35). Blood was processed by the CellSieve™ microfiltration technique at 5 institutions and stained for cytokeratin 8, 18 & 19, EpCAM, and CD45. After identification/imaging, the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used to quench fluorescence signal of cells and then restain with vimentin/CD146/CD144, CD14/CD11b/CD41, CD11c/CD68/TIE2, or CD34/CD41/CD61. After staining, QUAS-R was again used to remove the fluorescence and samples restained with a third panel. Each patient sample was stained, quenched and restained with the above mentioned panels. Results: In agreement with a number of studies, CAMLs were found in 86% of cancer patients (n=131/152), with increased detection from stage 1 (71%), followed by stage 2 (94%), stage 3 (88%) to stage 4 (88%). Breast cancer had the most CAMLs per sample (14.1 cells/7.5mL), followed by prostate (6.8), renal cell carcinoma (4.9) and lung (3.2). CD34 was most prevalent, found on 88% of CAMLs, followed by cytokeratin (81%), CD41 (79%), CD61 (78%), CD45 (75%), CD14 (72%), vimentin (63%), EpCAM (56%), CD146 (53%), CD68 (44%), CD11c (38%), TIE-2 (25%) and CD11b (0%). Based on our results, CAMLs seem to express overlapping phenotypes from a variety of lineages i.e. macrophage (CD14/CD68/CD11c), epithelial (cytokeratin/EpCAM), endothelial (CD146/TIE-2) and megakaryocyte (CD41/CD61). Conclusions: Although identification of CAMLs is straightforward by morphological criteria (size and nuclear profile), their identification and lineage remains undetermined. We report the first mass screening of CAMLs to determine phenotypic expression. These data suggest that CAMLs cannot be grouped into any known cell subtype based on their expression profiles and represent a heterogeneous and variably differentiated population whose biological consequences in cancer remain under investigation. Citation Format: Daniel L. Adams, R Katherine Alpaugh, Thai H. Ho, Steven H. Lin, Jeffrey R. Marks, Raymond Bergan, Stuart S. Martin, Saranya Chumsri, Cha-Mei Tang, Massimo Cristofanilli. Multiplex phenotyping of circulating cancer associated macrophage-like cells in patients with solid tumors [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 3798. doi:10.1158/1538-7445.AM2017-3798

Abstract Background: Blood-based testing can be used as a noninvasive method to recover and analyze circulating tumor-derived cells for clinical use. Circulating cancer-associated macrophage-like cells (CAML) are specialized myeloid cells found in peripheral blood and associated with the presence of solid malignancies. We measured CAMLs prospectively in peripheral blood to ascertain their prevalence, specificity, and sensitivity in relation to breast disease status at clinical presentation. Methods: We report on two related but separate studies: 1) CellSieve microfilters were used to isolate CAMLs from blood samples of patients with known malignant disease (n = 41). Prevalence and specificity was compared against healthy volunteers (n = 16). 2) A follow-up double-blind pilot study was conducted on women (n = 41) undergoing core-needle biopsy to diagnose suspicious breast masses. Results: CAMLs were found in 93% of known malignant patients (n = 38/41), averaging 19.4 cells per sample, but none in the healthy controls. In subjects undergoing core biopsy for initial diagnosis, CAMLs were found in 88% of subjects with invasive carcinoma (n = 15/17) and 26% with benign breast conditions (n = 5/19). Conclusion: These preliminary pilot studies suggest that the presence of CAMLs may differentiate patients with malignant disease, benign breast conditions, and healthy individuals. Impact: We supply evidence that this previously unidentified circulating stromal cell may have utility as a screening tool to detect breast cancer in various malignancies, irrespective of disease stage. Cancer Epidemiol Biomarkers Prev; 25(7); 1037–42. ©2016 AACR.

An all-in-one Circulating Tumor Cell (CTC) isolation platform with streamlined multiplex integration of proteomic and genomic clinical testing.

Abstract Background: Blood-based biopsies can be used as a non-invasive method to recover both Circulating Tumor Cells (CTCs) and Circulating Cancer Associated Macrophage-Like cells (CAMLs) from the blood of cancer patients. CAMLs are a newly-defined circulating immune cell type, described as a subtype of circulating stromal cells, known to engulf tumor cells/debris. CAMLs are identified by their large size, 30-300 microns, and aberrant nuclear structure. We studied the peripheral blood of cancer patients to ascertain the prevalence, specificity and sensitivity of CAMLs in relation to their disease status at presentation, including a variety of benign and malignant diseases. We supply evidence that this previously unidentified circulating cell can be used as a screening tool to detect solid tumors in numerous malignancy subtypes in all disease stages. Methods: CellSieveTM microfilters were used to isolate CAMLs from 7.5 mL of whole peripheral blood. The pore size of CellSieveTM is 7 microns, capable of isolating both CTCs and CAMLs based on size. Collected cells were fixed, permeabilized, and stained with DAPI and antibodies against cytokeratin 8, 18 and 19, CD14, and CD45. CAMLs were defined as enlarged, multinuclear cells with diffuse cytoplasmic cytokeratin staining, and/or CD14+, and/or CD45+. CTCs were defined as filamentous cytokeratin cells that are CD45-. Study 1: Blinded peripheral blood samples from 61 cancer patients were tested, including Stage I-IV breast, pancreatic, lung and prostate cancers, and newly diagnosed, untreated patients. Non-blinded blood samples from 30 healthy volunteers served as controls. Study 2: Double blinded peripheral blood samples from 20 patients with abnormal mammogram results were drawn at time of tissue biopsy and screened. After analysis, unblinding showed a distribution of malignant breast disease (n = 10), benign (n = 8), or atypical hyperplasia (AH) (n = 2). Results: Study 1: CAMLs were found in 94% patients with Stage III/IV cancers, 87% with Stage I/II cancers, and 89% of all patients, regardless of cancer type. Specifically, CAMLs were found in 77% of prostate, 100% of pancreatic, 80% of lung, and 100% of breast patient samples. CAMLs were not found in healthy control samples (sensitivity: 84-96%, Specificity: 100%, PPV: 100%, NPV: 88-97%). Study 2: CAMLs were found in 8 patients with malignant disease, 2 with benign disease, and 2 with AH (sensitivity: 80%, Specificity: 75%, PPV: 80%, NPV: 75%) Conclusions: We present evidence that CAMLs can be used as a non-invasive blood based biopsy, to detect the anatomical presence of solid malignancies and potentially pre-malignant lesions. The presence of CAMLs in newly diagnosed patient samples suggests their use as a blood biomarker for early stage cancer screening in a broad population. Preliminary data from a double blind breast cancer study suggests that CAMLs may differentiate between benign and malignant breast diseases. Citation Format: Daniel Adams, Katherine Alpaugh, Massimo Cristofanilli, Stuart Martin, Saranya Chumsri, Raymond C. Bergen, Susan Tsai, Martin Edelman, Peixuan Zhu, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang, Jeffrey R. Marks. Cancer associated macrophage-like cells as a blood-based biomarker for the screening of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5165. doi:10.1158/1538-7445.AM2015-5165

AbstractRecent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch® CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45−/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45− cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45− cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45−/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non‐EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.