Alpaugh, R. Katherine

Abstract Background: We previously demonstrated that cancer associated macrophage-like cells (CAMLs) are cancer specific giant polyploid cells circulating in the blood of patients with solid tumors. Building on our initial discovery, others have shown that these hyperploidy cells are an innate immune response that is associated with decreased survival. However to date, no studies have been done to elucidate their clinical significance as in relation to the various stages of malignant disease. We established a 4 year prospective study of 315 patients from a variety of solid tumors (breast, prostate, lung, renal cell, pancreas, and esophageal) comparing the morphological properties of CAMLs in both early and late stage disease as they relate to progression free and overall survival (PFS/OS). These data suggest that CAML size appears have a strong negative correlation with progression and survival of cancer patients, irregardless of stage of disease, indicating their possible use as a non-invasive blood based biomarker in solid tumors. Methods: A prospective multi-institutional study used anonymized peripheral blood from 315 cancer patients [stage I (n=62), stage II (n=72), stage III (n=69) & stage IV (n=106)] from subjects with breast (n=59), esophageal (n=27), lung (n=59), renal cell carcinoma (n=37), prostate (n=74), pancreas cancers (n=59). CAMLs were isolated by the CellSieve™ microfiltration technique at 5 institutions and stained for cytokeratin 8, 18, & 19, CD14 and CD45. After imaging, a size based threshold ≥50µm was used to separate the patient cohorts, based on previously published assessments. Results: At least one CAML was found in 92% of cancer patients (n=289/315), with the lowest sensitivity in stage 1 (86%), followed by stage 2 (90%), stage 3 (97%) and stage 4 (95%). The property of CAML size was then evaluated. Overall, CAMLs of <50µm in size had superior PFS (HR: 3.8; 95% CI 2.8-5.3; p<0.001) and OS (HR=3.8; 95% CI 2.6-5.7;p<0.001) than those with ≥50 µm CAMLs. By each stage individually, stage 1 (PFS HR=7.5; 95% CI 3.2-17.7; p<0.001), stage II (PFS; HR=4.1; 95% CI 1.9-8.8; p<0.001), stage III (PFS HR=3.1; 95% CI 1.7-5.7; p=0.007), and stage IV (PFS HR=2.7; 95% CI 1.6-4.4; p<0.001). Conclusions: In this first large scale prospective study of the clinical utility of CAMLs, it appears that they are present in every stages of invasive solid tumor malignancies, with prevalence increasing with stage. Interestingly decreased CAML size was found to be associated with improved outcome and longer PFS. These data suggest that CAML detection and size assessment constitute a new real time predictor of progression, and survival in both early and late stage disease indicating the need for additional validation studies. Citation Format: Daniel L. Adams, Steven H. Lin, R. Katherine Alpaugh, Thai Ho, Jeffrey R. Marks, Stuart S. Martin, Susan Tsai, Saranya Chumsri, Cha-Mei Tang, Massimo Cristofanilli, Raymond Bergan. Cancer associated macrophage-like (CAMLs) cells in blood predict progression and survival for all stages of solid tumors [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 2631.

Abstract Background: Tumor endothelial cells (ECs) are a population of stromal cells required for tumor growth that cooperate with tumors to form angiogenic structures. In blood, circulating ECs (CECs) are normal constituents of healthy individuals, although a Cancer Associated Vascular Endothelial cell (CAVE) subtype has been observed in cancer patients. The CAVE population has been isolated and identified using their large size or multicellular clustering and a pooled mixture of classical EC markers (i.e. CD31 and CD146). However, there has been no attempt to differentiate CAVEs from the many EC subtypes. This is not surprising as in-depth phenotyping of ECs requires an array of biomarkers that until recently has not been feasible. A multi-phenotypic screening of EC markers was tested on CAVEs from 116 blood samples in 3 types of solid tumors. This data suggests that CAVEs exist as a common and diverse subtype of tumor derived CECs that may express cytokeratin (CK) and various EC biomarkers, correlating to disease stage. Methods: Peripheral blood samples from 116 cancer patients (stage I-IV) were drawn from 2012-2014 including breast (n=42), lung (n=39) and prostate (n=35), as well as blood from 34 healthy controls. Blood was processed by an established filtration approach, i.e. the CellSieveTM microfiltration technique (Creatv MicroTech), filtering blood by size exclusion and staining cells for CK 8, 18 & 19, EpCAM and CD45. After identification and imaging, the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used to remove fluorescence signal and restain all cells with CD31, CD146, CD144, & DAPI. After reimaging, QUAS-R was again used to remove fluorescence and restain the cells for CD14, CD105, CD34, & DAPI. Results: Out of 116 patient samples, we identified CAVEs in 63 patients (54%) based on positivity of CD31, CD144 or CD146, but none were found in healthy controls. CAVEs per 7.5mL sample in patients averaged 5.1 (breast), 5.6 (prostate) and 7.9 (lung). Presence of CAVEs appeared related to stage with 26% in stage 1, 61% in stage 2, 68% in stage 3, and 74% in stage 4 patients. No CAVEs were positive for CD14 or CD45. CD31 was the most present marker, found on 93% of CAVEs, followed by CD144 (85%), CD34 (64%), CD146 (45%), & CD105 (4%). In contrast with the previous study on this topic, CK was found in 67% of CAVEs, but was not a universal marker. Conclusions: It has been reported that CK+ and CD45- CECs are isolated from the blood of cancer patients in colon and lung cancers, prompting some to classify them as circulating tumor cells. However, subtyping these CECs is incomplete when characterized with only 3-4 biomarkers. A multi-phenotypic subtyping technique was used to properly identify and subtype these CECs in cancer patients. This data suggest that a subset of CECs, e.g. CAVEs, are found in circulation as CK+/CD45-, but exist as a heterogeneous population of cancer specific circulating cells that require further study. Citation Format: Daniel L. Adams, R. Katherine Alpaugh, Steven H. Lin, Jeffrey R. Marks, Raymond Bergan, Stuart S. Martin, Sarany Chumsri, Massimo Cristofanilli, Cha-Mei Tang, Steingrimur Stefansson. Identifying, subtyping and classifying tumor associated circulating endothelial 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 778. doi:10.1158/1538-7445.AM2017-778

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.

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.