Citations

Abstract Background: Activation of the RAS/MAPK pathway is associated with reduced tumor-infiltrating lymphocytes and poor outcomes in triple-negative breast cancer (TNBC). This trial evaluated the efficacy of pembrolizumab and MEK inhibitor, binimetinib. Here we evaluated potential biomarkers in peripheral blood to predict response. Methods: Patients with unresectable locally advanced or metastatic TNBC with ≤ 3 prior lines of therapy were enrolled. Treatment includes a 2-week run-in with binimetinib followed by pembrolizumab. There were 2 dose levels (DL) with binimetinib at 45 mg at DL 0 and 30 mg at DL -1. A standard 3+3 design was used in phase I, and Simon’s two-stage Optimal design was used in phase II. Circulating tumor cells (CTC) and circulating cancer-associated macrophage-like cells (CAML) were isolated using CellSieve microfilters and immunofluorescently labeled with PD-L1 and p-ERK. Wilcoxon rank sum test and Cox regression model were used for analysis. Results: A total of 22 patients were enrolled, with a median age of 58 years old. Dose-limiting toxicity (DLT) was observed in 2 out of 4 patients in DL 0, with grade 3 ALT abnormality, flank pain, and nausea. In the next 6 patients in DL -1, there was 1 DLT with grade 3 AST/ALT abnormality. There were 17 patients treated with DL -1 and were evaluable for response. The objective response rate (ORR) was 29.41% (95% CI: 10.31-55.9) with 1 complete response (CR) and 4 partial responses (PR). The clinical benefit rate (CBR ≥ 24 weeks) was 35.29% (95% CI: 14.21-61.67). ORR in patients without liver metastases was 55.56% (95% CI: 21.20 - 86.30), and CBR was 66.67% (95% CI: 29.93-92.51). There was no response observed in all 5 patients with liver metastases. Baseline mean CTC count was 1.3 cells, and CAML count was 8.9 cells/7.5 mL. Baseline PD-L1 in CAML (p 0.04) and decline in CAML size (p 0.02) after 1 cycle were significantly associated with CBR. However, baseline CTC count, CAML count (p 0.64), CAML size (p 0.46), p-ERK in CAML (p 0.23), and changes in CTC count, CAML count (p 0.83), p-ERK (p 0.07), and PD-L1 (p 0.08) in CAML were not significantly associated with responses. Using Cox regression analysis, a reduction in CAML count (p 0.02), CAML size (p 0.01), and PD-L1 in CAML (p 0.03) were associated with significant improvement in overall survival but not the reduction in p-ERK (p 0.6). Conclusions: Pembrolizumab and binimetinib at 30 mg are safe with manageable toxicities. Promising activity was observed in patients without liver metastases. Baseline PD-L1 expression, early reduction in CAML count, size, and PD-L1 expression were significantly associated with subsequent responses, providing potential noninvasive biomarkers to predict response to this combination. Future larger clinical trials are warranted to further evaluate the efficacy of this combination. Citation Format: Saranya Chumsri, Joseph J. Larson, Daniel L. Adams, Kathleen S. Tenner, Cha-Mei Tang, Morgan T. Weidner, Amanda N. Arnold, Dana L. Haley, Pooja Advani, Kostandinos Sideras, Alvaro Moreno-aspitia, Edith A. Perez, Keith L. Knutson. Peripheral blood biomarkers in Phase II study of pembrolizumab in combination with oral binimetinib in patients with unresectable locally advanced or metastatic triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5454.

3054 Background: Tumor Macrophage Fusion Cells (TMFCs) are hybrids fusions of tumor cells and macrophage/immune cells that have recently been documented in advanced solid tumors, such as metastatic Breast Cancer (mBC). Further, TMFCs are found in the blood of patients (pts) as a CD45+/CD14+ binucleated subtype of standard circulating tumor cells (CTCs), which are Cytokeratin+ (CK+) & CD45 negative, but distinct from highly hyperploidy cells known as cancer associated macrophage-like cells. However, no study has explored the clinical meaning of TMFCs in blood. To better elucidate the clinical meaning of TMFCs, we prospectively pooled 137 mBC pt samples prior to induction of new therapy, ie baseline (BL), and after therapy induction (FU). Both TMFCs or standard CTCs were identified and enumerated to analyze their prognostic value for progression-free survival (PFS) and overall survival (OS). Methods: In this prospective study, we collected 7.5 mL blood samples from n=137 mBC pts enrolled in prospective trials from multiple institutions before starting new treatment lines for newly progressive mBC. If possible, an optional FU sample was collected (n=73) after BL (median=4.9 weeks). TMFCs & CTCs were isolated using a CellSieve microfilter and differentiated by staining for CK, CD45, CD14, and DAPI. Cox proportional regression hazard ratios (HRs) with 95% Confidence Intervals (CI) for PFS & OS by univariate & multivariate analyses were based on RECIST v1.1, determined by local institutional pathologist over 24 months (M). Results: TMFCs were detected in 26.3% (n=36/137) at BL and associated with significantly worse PFS (HR=3.3, CI 1.9-5.7, p<0.001) & OS (HR=2.7, CI 1.4-5.1, p=0.005). TMFCs were also detected in 27.4% (n=20/73) at FU (n=73) and prognostic for PFS (HR=5.2, CI 2.3-11.9, p<0.001) & OS (HR=5.1, 1.8-14.2, p=0.004). Further, pts with increases in TMFCs between BL and FU had worse PFS (HR=4.3, CI 1.9-10.1, p=0.001) & OS (HR=4.5, CI 1.6-12.7, p=0.011). In contrast, pts with CTCs were found in 39.4% (n=54/137) at BL, which were prognostic for worse PFS (HR=2.0, CI 1.3-3.2, p=0.005) and not prognostic for OS (HR=1.7, CI 0.9-3.0, p=0.103). At FU, pts with CTCs were found in 38.4% (n=28/73), which were prognostic for worse PFS (HR=3.6, CI 1.8-7.3, p=0.001) & OS (HR=3.2, CI 1.3-8.0, p=0.021). Overall at BL, pts without CTCs or TMFCs had a 5.7 M mPFS & >24M mOS, CTCs alone had a 5.0 M mPFS & 20.3 M mOS, TMFCs had 2.0 M mPFS & 5.8 M mOS. At FU, pts without CTCs or TMFCs had a 5.8 M mPFS & 14.5 M mOS, CTCs alone had a 3.3 M mPFS & 14.6 M mOS, while TMFCs pts had a 1.7 M mPFS & 4.8 M mOS. Conclusions: TMFCs appear to represent a CTC subgroup with additional prognostic value to standard CTCs, that may correlate with significantly faster PFS and OS versus pts with CTCs alone, or without CTCs. Subtyping treatment regimens is ongoing to determine if the TMFC populations are responsive to specific therapy types in these mBC pts.

e14548 Background: Micronuclei (MN) are small, excised DNA fragments resulting from biological DNA repair mechanisms in response to internal chromosomal instability, such as those caused by genotoxic events (i.e. chemotherapy and radiation), and whose formation in tumor stromal cells in blood circulation is prognostic for worse clinical outcomes (Fenech 2020). Further, mutagenic events occurring during the formation of MN are associated with the development of cancer neoantigens, which are a major element in the programmed cell death ligand (PD-L1) pathway that influences response to immunotherapies. We hypothesized that MN can be detected in metastatic breast cancer (mBC) patients (pts) within circulating stromal cells (CStCs) that prognosticates for clinical outcomes and may associate with PD-L1 cell expression. Methods: We initiated a prospective pilot study using a compiled set of n=124 mBC samples, from multiple institutions, enrolled prior to starting new lines of treatment for progressive mBC. We enumerated MN formation found in CStCs from whole peripheral blood (7.5mL), filtered for CStCs, then stained for PD-L1 and DAPI. DAPI was used to identify MN, defined by small (<3 µm) DAPI+ circular formations within the cytoplasm, separate from primary nuclei. In conjunction, all cells were stained with PD-L1 with expression normalized to background and evaluated against MN by T-test analysis. MN presence was compared to all available clinical variables, including induction of PD-L1 immunotherapy. Pts’ progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate and multivariate analyses based on RECIST v1.1 over 24 months. Results: MN were identified in 56% (n=70/124) of CStCs and their presence was significantly prognostic for worse PFS (HR=2.4, CI= 1.53-3.76, p<0.001) & OS (HR= 1.89 CI=1.07-3.34, p=0.039) over 24 months. Overall, mPFS of pts with MN was 2.8 months and without MN was 12.1 months. The mOS of pts with MN was 12.7 months and without MN was 20.8 months. Additionally, MN positive CStCs had a significantly higher PD-L1 expression than MN negative CStCs (p<0.001), with 67% of MN positive pts (n=46/66) having high PD-L1 expression while 30% of MN positive pts (n=20/66) having low PD-L1. Further, evaluation of PD-L1 in CStCs showed a linear relationship (R=0.9806) between MN number and PD-L1 in cells, with 0 MN average=0.016, 1 MN=0.200, 2 MN=0.318, and ≥3 MN=0.400. Conclusions: These initial findings suggest that MN are detected in pts with advanced mBC and may identify pts with faster progression and worse survival outcomes. Further, the number of MN in CStCs appears to correlate with higher PD-L1 expression within cells. Follow up clinical information and subtyping studies are ongoing to also evaluate of the effect of MN positive CStCs on PD-L1 immunotherapies in these pts.

Abstract Background: Micronuclei (MN) are a result of biological DNA repair mechanisms which form due to internal chromosomal aberrations indicating sub-clonal cancer populations with higher cell survivability and drug therapy resistance. MN are often observed as small fragments of nucleic acids excised from a primary nucleus in Circulating Stomal Cells (CStCs) in response to DNA damage. CStCs with damaged DNA undergoing repair mechanisms, such as those that form MN, are likely to have upregulated expression of programmed cell death ligand (PD-L1) which can be abnormally high on cancer cells that have high mutational burdens. We evaluated CStCs in metastatic breast cancer (mBC) patients for presence of MN and the cell's PD-L1 expression, to determine its prognostic significance to clinical outcomes. Methods: We enumerated MN formation in CStCs in a prospective pilot study using n=76 mBC patients starting new lines of treatment. Whole peripheral blood (7.5mL) was procured and filtered for CStCs and then stained for PD-L1. DAPI was used to identify MN, defined by small (&lt;3 µm) DAPI+ circular formations within the cytoplasm, separate from the primary nucleus. We compared number of MN to PD-L1 expression of all cells, and the MN presence to all available clinical variables. Patients’ progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate analysis based on RECIST v1.1 over two-years. Results: MN were identified in 62% (n=46/76) of CStCs and MN positive CStCs had a significantly higher PD-L1 expression than MN negative CStCs (p=0.0082). A linear relationship between increasing MN number and higher PDL-1 expression within cells was identified, R=0.9411. Further, presence of MN with in CStCs was significantly prognostic for worse PFS (HR=2.62 95%CI 1.50-4.59, p=0.0012) and worse OS (HR= 3.07 95%CI 1.56-6.03, p = 0.002) over 24 months. Conclusion: MN formations in mBC are observable biomarkers of an underlying repair mechanism which suggest the survivability of sub-clonal cancer populations associated with more aggressive cancer subtypes with worsening progression rates. Further studies to evaluate the effect of PD-1 immunotherapies in MN positive patients is ongoing. Citation Format: Dimpal M. Kasabwala, Massimo Cristofanilli, R. Katherine Alpaugh, Saranya Chumsri, Carolina Reduzzi, Cha-Mei Tang, Rena G. Lapidus, Giuseppe Del Priore, William V. Williams, Daniel L. Adams. Micronuclei in circulating stromal cells correlates with PD-L1 expression and predicts progression in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 304.

The usage of beta blockers in breast cancer (BC) patients is implicated in the reduction in distant metastases, cancer recurrence, and cancer mortality. Studies suggest that the adrenergic pathway is directly involved in sympathetic-driven hematopoietic activation of pro-tumor microenvironmental proliferation and tumor cell trafficking into the circulation. Cancer-associated macrophage-like cells (CAMLs) are pro-tumor polynucleated monocytic cells of hematopoietic origin emanating from tumors which may aid in circulating tumor cell (CTC) dissemination into the circulation. We examined the linkage between Beta-2 adrenergic receptor (B2AR) signaling in CAMLs and CTCs by establishing expression profiles in a model BC cell line (MDA-MB-231). We compared the model to CAMLs and CTCs found in patents. Although internalization events were observed in patients, differences were found in the expression of B2AR between the tumor cell lines and the CAMLs found in patients. High B2AR expression on patients’ CAMLs was correlated with significantly more CAMLs in the circulation (p = 0.0093), but CTCs had no numerical relationship (p = 0.1565). High B2AR CAML expression was also significantly associated with a larger size of CAMLs (p = 0.0073), as well as being significantly associated with shorter progression-free survival (p = 0.0097) and overall survival (p = 0.0265). These data suggest that B2AR expression on CAMLs is closely related to the activation, intravasation, and growth of CAMLs in the circulation.

3056 Background: In metastatic Breast Cancer (mBC), Circulating Tumor Cells (CTCs) are established prognostic indicators of patients (pts) not responding to new lines of therapy and who have poor clinical outcomes. However, CTCs are typically found in < 20% of mBC pts and many pts without CTCs will also progress. Recently an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in > 90% of mBC pts and were also indicative of poor clinical outcomes. As CTCs & CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for therapy response, we evaluated CTCs & CAMLs before and after initiation of new therapies in mBC to determine their combined prognostic/predictive values. Methods: An observational prospective 2-year multi-institutional study was undertaken to evaluate CTCs & CAMLs before, and after, induction of any new line of therapy in pts with diagnosed mBC (n = 101). Anonymized and blinded blood samples were taken at baseline (BL), prior to starting a new systemic therapy, and a 2nd sample (T1) taken after therapy initiation (̃30 days). Blood was filtered by CellSieve filtration. The quantities and subtypes of CTCs & CAMLs were analyzed based on RECIST v1.1 for progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) by censored univariate & multivariate analysis at 2 years. Results: CTCs were identified in 35% (n = 35/101) of pts at BL & 24% (n = 24/101) at T1, with a single CTC at T1 being highly prognostic for worse PFS HR = 6.2 95%CI 3.0-13.2, p < 0.001 & OS (HR = 5.1 95%CI 2.0-13.4, p = 0.002. In parallel, CAMLs were found in 93% of BL and 86% of T1 samples, and whose decreases were significantly prognostic for improved PFS (HR = 2.7, 95%CI 1.4-5.1, p = 0.006) and OS (HR = 4.4 95%CI 1.5-13.2, p = 0.018) when CTCs were absent. Overall ≥1 CTC at T1 (n = 24) had median PFS = 2.4 & mOS = 4.8 months (mos), however, in pts without CTCs plus an increase in CAMLs (n = 36) had mPFS = 5.9 & mOS = 14.1 mos, while in pts without CTCs plus a decrease in CAMLs (n = 41) had mPFS = 14.8 & mOS = 18.8 mos. Conclusions: Our data confirms that pts with persistent CTCs have the worst clinical outcomes. Further, simultaneous CAML quantification provided a new dynamic predictive blood based biomarker in pts without detectable CTCs which may be useful to better individualize therapy and improve outcomes, though future studies are need to validate these findings.

Abstract Background: CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and endocytosis may identify highly aggressive breast cancer (BC) subtypes likely to spread via the circulatory system. We first studied the activation and endocytosis of CCR5 in response to its ligand CCL5 (RANTES) in the model BC cell line MDA-MB231. We then screened two types of circulating tumor-associated cells (TACs) with known negative clinical outcomes, 1) circulating tumor cells (CTCs) and 2) cancer-associated macrophage-like (CAMLs) cells, to evaluate CCR5 upregulation in relation to disease progression in metastatic breast cancer (mBC). Methods: MB231 cells were used to visualize CCR5 activation within the cell's surface and intracellular space using an anti-CCR5 antibody after stimulation with RANTES. MB231 cells were stained with LAMP-1A to verify co-localization of CCR5 and RANTES to endosomes. Anonymized peripheral blood samples from (n=54) mBC patients were obtained in accordance with local IRB regulations and with informed consent. TACs were isolated using a low-flow CellSieve Microfiltration system and stained for Cytokeratin, CD45, and CCR5. CCR5 expression signal and localization were evaluated on all TACs via fluorescence microscopy. Results: CCR5 in MB231 cells appeared as ~1μm clusters known as “CCR5 pools”, which can be found on the cell's surface, within the cytoplasm, and in the nuclear space. CCR5 pools were upregulated in the presence of RANTES, which appears to relate to endocytosis and nuclear translocation. Within the TACs of mBCs, we found similar patterns of CCR5 pooling with 70% of patients (n=38) having CCR5+ CAMLs and 41% (n=22) having CCR5+ CTCs. It was found that higher numbers of CCR5 pools (≥10pools/cell) were correlated to a 2-fold increase in both CTCs and CAMLs in circulation. In the analysis of outcome data, it was determined that patients having ≥10 CCR5 pools on their CAMLs was a highly significant predictor of worse progression free survival (PFS) and overall survival (OS) (PFS HR=4.8, CI 95% 1.9-11.6, p=0.0015) and (OS HR=3.6, CI 95% 1.3-10.0, p=0.0331). In CTCs ≥10 pools/cell was not a significant predictor for worse PFS and OS. However, ≥5 CCR5 pools/cell significantly predicted for worse PFS but not OS (PFS HR=2.9, CI 95% 1.2-7.4, p=0.0418 and OS HR=3.3, CI 95% 1.1-9.3, p= 0.0539). Conclusion: We observed the activation and endocytosis of CCR5 pools using RANTES in MD-MB231 cell lines, and then demonstrated that this upregulated motility pathway appears to be conserved in migratory cells in mBC patients. Further, this upregulation of CCR5 appears to correlate with worse clinical outcomes, which may provide an explanation for how tumor-associated cells move into circulation and acquire metastatic potential. Citation Format: Ashvathi Raghavakaimal, Massimo Cristofanilli, Cha-Mei Tang, R. K. Alpaugh, Kirby P. Gardener, Saranya Chumsri, Daniel L. Adams. CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 589.

Abstract Background: Prostate cancers (PC) are intrinsically complex, with few prognostic biomarkers that differentiate aggressive and indolent disease. Cancer associated macrophage like cells (CAMLs) have shown to be significant independent prognostic indicators for poor survival in tumors if engorged to ≥50μm. However, no group has examined CAMLs' in PC. In this prospective pilot study, we analyzed PSA & ≥50μm CAML presence in (n=51) Stage I-III & (n=41) metastatic PC (mPC) prior to the start of new treatments to examine CAMLs' on outcomes. We found that ≥50μm CAML presence significantly predicted worse patient survival in mPC. These preliminary results suggest that CAMLs may serve as a cheap, non-invasive prognostic biomarker that predicts for worse outcomes in both local & mPC prior to new therapies. Methods: We prospectively recruited (n=92) PC patients in this pilot study to examine CAML's prognostic significance in non-metastatic (stages I-III) and mPC. Of (n=92) total patients, 15% (n=14/92) were stage I, 32% (n=29/92) stage II, 9% (n=8/92) stage III, and 45% (n=41/92) Stage IV. Prior to the induction of new therapy or first line therapy for newly diagnosed patients, 7.5mL peripheral baseline (BL) blood was collected. Blood was filtered via CellSieveTM microfiltration, which excludes cells by size ≥7μm. In parallel, changes in PSA was monitored to compare prognostic significance against ≥50μm CAMLs. Wilcoxon univariate analysis was used to analyze ≥50μm CAMLs and PSA in progression free survival (PFS) and overall survival (OS). Results: CAML's were identified in 78.9% (n=71/90) of available BL samples, with two samples failing from clotting. Stage I-III patients had an average of ~3 CAMLs/7.5mL blood, whereas mPCs contained ~6 CAMLs/7.5mL. Average CAML size increased linearly with advancing stage of disease with stage I patients averaging 23μm, stage II 33μm, stage III 65μm, and stage IV 78μm. Of available BL samples, 41% (n=38/90) patients with ≥50μm CAMLs significantly predicted for worse PFS (HR=10.0, p&lt;0.001, 95%CI=0.07-0.31) and OS (HR=34.2, p&lt;0.001, 95%CI=0.03-0.21). Further, ≥50μm CAMLs significantly predicted for poorer survival in mPC with PFS (HR=9.4, p=0.028, 95%CI=0.18-0.84) and OS (HR=5.1, p=0.003, 95%CI=0.07-0.53), but only non-significantly trended in non-metastatic disease. Initial analysis of PSA found that high PSA at BL was a significant predictor for worse outcomes in patients, while changes in PSA was not a predictor. Conclusions: CAMLs have shown potential as a predictive non-invasive blood based biomarker across multiple solid malignancies, and here we compared this biomarker in PC. While preliminary, these data suggests that engorged CAML presence prior to the start of new treatment may be a statistically significant predictor of survival in both the local and metastatic setting, and a larger validation cohort should be established. Citation Format: Daniel J. Gironda, Raymond Bergan, R K. Alpaugh, Daniel C. Danila, Tuan L. Chuang, Brenda Y. Hurtado, Thai Ho, Cha-Mei Tang, Daniel L. Adams. Cancer associated macrophage like cells predict aggressive disease in local and metastatic prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 357.

3039 Background: Patients with multiple organ metastases have poorer prognoses than those with a single organ metastasis, are frequently associated with drug resistance, and have higher tumor burden. Engorged (≥50um) Cancer Associated Macrophage-Like Cells (CAMLs) are a circulating stromal cell subtype detected in the blood of patients with solid tumors at high risk for recurrence or progression. While numerous studies have shown that ≥50um CAMLs predict poor clinical outcomes, meta-analysis of these studies have also suggested that hyper engorged CAMLs ≥100um (heCAMLs) may be associated with multifocal metastatic disease and even worse outcomes. In this prospective study, we evaluated the presence of heCAMLs in patients with metastatic disease and demonstrated a strong relationship with multi organ spread, which also correlated with shorter Progression Free Survival (PFS) and Overall Survival (OS). Methods: We prospectively recruited 151 patients with metastatic (m) mbreast (n = 58), mlung (n = 34), mprostate (n = 39), and mrenal (n = 20) cancers. Peripheral blood was collected prior to the induction of new treatment for metastatic cancer. Cells were isolated following standard CellSieve techniques, then imaged and measured in ZenBlue. Multi organ metastasis was defined as spread to ≥2 distant organ sites, or any spread to the brain. Single factor ANOVA was conducted to compare heCAML presence in multi organ metastatic patients versus patients with single organ site metastasis. Univariate and multivariate analysis was run to evaluate for PFS and OS against heCAMLs, and all known clinical parameters. Results: 150 viable samples (excluding 1 failed sample) were obtained. Multi organ metastases were present in 55% (n = 83/150) of patients. heCAMLs were found in 59% (n = 49/83) of the multi organ metastatic population, but only in 16% (n = 11/67) of the single site metastatic cohort (p < 0.001). heCAML presence appeared to differentiate multi organ vs single organ metastases in mbreast (85% vs. 52%, p = 0.006), mlung (71% vs. 26%, p = 0.025), mprostate (75% vs. 37%, p = 0.029), and mRCC (88% vs. 36%, p = 0.025). Further, in all n = 150 patients, heCAML presence predicted a significantly shorter median PFS of 4.5 versus 7.2 months, 24 month PFS (HR = 1.67, 95%CI = 1.13-2.45, p = 0.013), and significantly shorter median OS of 13.1 versus 20.4 months, 24 month OS (HR = 2.05, 95%CI = 1.24-3.39, p = 0.008). Conclusions: We examined a non-invasive prognostic blood based assay to determine its relationship to multi organ metastatic spread as well as its prognostic value in several solid cancers. These results showed patients with heCAMLs had higher rates of multi organ metastases, and appear to predict for shorter PFS and OS. Studies of larger cohorts are needed for prospective validation of these initial findings.

3535 Background: Peripheral blood allows for a simple non-invasive method for isolating various cancer associated circulating stromal cells (CStCs) which may predict for cancer presence. Cancer Associated Macrophage-Like cells (CAMLs), a specific CStC, are phagocytic myeloid cells that derive from an immunological response to cancer and emanate from primary tumors. Using a filtration platform we screened the peripheral blood of untreated newly diagnosed cancer patients (n = 308) for CAMLs. In parallel, we screened patients with newly diagnosed non-malignant diseases, i.e. lupus, benign cysts, etc. (n = 39), and healthy control samples (n = 76). We found that CAMLs are highly prevalent (87%) in the blood of cancer patients, but uncommon in non-malignant conditions (20%) & absent in healthy individuals (0%). Methods: Anonymized peripheral blood were taken from 308 cancer patients after confirmation of invasive malignancy [stage I (n = 76), stage II (n = 73), stage III (n = 72), stage IV (n = 65) and unstaged non-metastatic (n = 22)] with pathologically confirmed lung (n = 65), pancreas (n = 53), breast (n = 52), prostate (n = 40), esophageal (n = 30), renal cell (n = 18), hepatocellular (n = 15), neuroblastoma (n = 10), melanoma (n = 8), and other (n = 17). Further, anonymized blood was taken from patients with untreated non-malignant conditions including benign breast masses (n = 19), lupus (n = 11), liver cirrhosis (n = 5), benign prostatic hyperplasia (BPH) (n = 3), and viral infection (n = 1); or from healthy control volunteers (n = 76). CAMLs were isolated from whole peripheral blood by the CellSieve™ microfiltration technique and defined as enlarged, multinuclear cells with cytokeratin and/or CD45/CD14 positive. Results: CAMLs were found in 87% of all cancer patients regardless of stage, ~5.4 CAMLs/7.5mL blood. Specifically, CAMLs were found in 80% of Stage I, 90% Stage II, 89% Stage III, and 97% Stage IV patients. No CAMLs were found in any healthy controls, but were found in 26% of benign breast masses, 18% of lupus, 0% of BPH and 0% of cirrhosis. In total, CAML sensitivity in cancer vs healthy was 87% (CI95% 82-90%), specificity = 100% (CI95% 95-100%), PPV = 100% (CI95% 100%), NPV = 67% (CI95% 58-71%). CAML sensitivity in cancer vs benign was 87% (CI95% 82-90%), specificity = 80% (CI95% 64-91%), PPV = 97% (CI95% 95-98%), NPV = 43% (CI95% 35-51%). Conclusions: CAMLs, a Circulating Stromal Cell subtype, is a sensitive blood based biomarker found in all stages of cancer that is rare in non-malignant conditions and absent in healthy individuals.