Alpaugh, R. Katherine

Abstract Background In 2008, bevacizumab received accelerated Food and Drug Administration (FDA) approval for use in human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC). Based on the preclinical and preliminary clinical activity of the trastuzumab and bevacizumab combination, ECOG-ACRIN E1105 trial was developed to determine if the addition of bevacizumab to a chemotherapy and trastuzumab combination for first-line therapy would improve progression-free survival (PFS) in patients with HER2-positive MBC. Findings: 96 patients were randomized to receive standard first-line chemotherapy and trastuzumab with or without bevacizumab between November 2007 and October 2009, and 93 began protocol therapy. Induction therapy was given for 24 weeks, followed by maintenance trastuzumab with or without bevacizumab. 60% (56/93) began carboplatin and 74% (69/93) completed 6 cycles of induction therapy. Primary endpoint was PFS. Median PFS was 11.1 and 13.8 months for placebo and bevacizumab arms, respectively (hazard ratio [HR] 95%, Confidence Interval [Cl] for bevacizumab vs. placebo: 0.73 [0.43–1.23], p = 0.24), and at a median follow-up of 70.7 months, median survival was 49.1 and 63 months (HR [95% Cl] for OS: 1.09 [0.61–1.97], p = 0.75). The most common toxicities across both arms were neutropenia and hypertension, with left ventricular systolic dysfunction, fatigue, and sensory neuropathy reported more frequently with bevacizumab. Conclusions In this trial, the addition of bevacizumab did not improve outcomes in patients with metastatic HER2-positive breast cancer. Although the trial was underpowered due to smaller than anticipated sample size, these findings corroborated other clinical trials during this time.

Abstract Background: Circulating tumor cell clusters (CTCCs) are aggregated groups of tumor cells that detached from primary tumors and circulate the bloodstream. However, while Circulating Tumor Cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined, with only a few descriptive investigations evaluating their clinical utility. Adding to CTCC complexity is that various subtypes exist, including homotypic clusters consisting of a homogenous population of tumor cells and heterotypic CTCCs made of CTCs with immune/stromal white blood cells (WBCs). Furthermore, CTCs can undergo Epithelial-Mesenchymal Transition (EMT), a process characterized by loss of epithelial traits and upregulation of mesenchymal traits, and can form clustered EMTs (CEMTs). Moreover, CTCs can fuse with macrophages forming Tumor Macrophage Hybrid Cells, aka Cancer-Associated Macrophage Like cells (CAMLs) when in circulation. We enumerated all these types of cells and complex aggregates in the peripheral blood of patients with metastatic breast cancer (mBC). Methods: We enumerated the 6 populations from patient samples enrolled in IRB aproved prospective pilot studies. Whole peripheral blood (7.5mL) was filtered and stained with cytokeratin (CK) & CD45/CD14 to identify CTCs. CTCs were defined as an intact DAPI nuclei and strong filamentous CK. Homotypic CTCCs were defined as ≥2 CTCs attached together. Heterotypic CTCCs were defined as ≥1 CTC attached to ≥1 WBC. EMTs were defined as having DAPI nuclei and weak non-filamentous CK. CEMTS were defined as ≥2 EMTs. CAMLs were defined as having an enlarged polynucleated DAPI, and positive for CD45/CD14 or non-filamentous CK. Median progression free survival (mPFS) and median overall survival (mOS) over 24 months (m) were evaluated. Results: We evaluated the blood of 79 mBC patients. Single CTCs were found in 57% of patients (n=34/79), homotypic CTCCs 15% (n=12/79), heterotypic CTCCs 66% (n=27/79), EMTs 56% (n=44/79), CEMTs 23% (n=18/79) and CAMLs 97% (n=77/79). Over 24 ms, patierns with detectable heterotypic CTCCs & homotypic CTCCs had the shortest response times at mPFS=2.8 & mOS=5.8 ms, or mPFS=2.1 & mOS=4.3 ms, respectively. This was followed by CTCs mPFS=3.7 & mOS=13.7 ms, EMTs mPFS=4.3 & mOS=14.4 ms, CAMLs mPFS=4.3 & mOS=14.4 ms, and CEMT mPFS=4.7 & mOS=13.2 ms. Additionally, CTCCs were rare in HER2+ patients at 4.4% (n=1/23), but no associations to other hormone statuses were found. Conclusion: CTCCs appear to represent an array of subtypes with different biological and clinical meanings. Here we enumerated CTCC subtypes in the blood of mBC patients and stratified the various types to clinical outcomes. These data suggest CTCCs are clinically important and further understanding of their biology in the context of tumor pathogenesis is needed. Citation Format: Daniel L. Adams, Carolina Reduzzi, Aravind Aryasomayajula, R. Katherine Alpaugh, Saranya Chumsri, Cha-Mei Tang, Giuseppe Del Priore, William V. Williams, Massimo Cristofanilli. Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2303.

Despite advancements in the early-stage detection and expansion of treatments for prostate cancer (PCa), patient mortality rates remain high in patients with aggressive disease and the overtreatment of indolent disease remains a major issue. Prostate-specific antigen (PSA), a standard PCa blood biomarker, is limited in its ability to differentiate disease subtypes resulting in the overtreatment of non-aggressive indolent disease. Here we assess engorged cancer-associated macrophage-like cells (CAMLs), a ≥50 µm, cancer-specific, polynucleated circulating cell type found in the blood of patients with PCa as a potential companion biomarker to PSA for patient risk stratification. We found that rising PSA is positively correlated with increasing CAML size (r = 0.307, p = 0.004) and number of CAMLs in circulation (r = 0.399, p < 0.001). Over a 2-year period, the presence of a single engorged CAML was associated with 20.9 times increased likelihood of progression (p = 0.016) in non-metastatic PCa, and 2.4 times likelihood of progression (p = 0.031) with 5.4 times likelihood of death (p < 0.001) in metastatic PCa. These preliminary data suggest that CAML cell monitoring, in combination with PSA, may aid in differentiating non-aggressive from aggressive PCas by adding biological information that complements traditional clinical biomarkers, thereby helping guide treatment strategies.

Abstract Background: Recently, it was described that macrophages and tumor cells can fuse to form tumor macrophage fusion cells (TMFs) which are detectable within primary tumors and patient’s (pts) circulation. However, there are multiple pathways and subsequent subtypes of TMFs, with limited data on the various TMF types, commonality in blood, and clinical relevance. Here we evaluated n=120 metastatic breast cancer (mBC) blood samples for CTCs & TMFs. We describe numerous types of TMFs with vastly different fusion phenotypes, including partial (i.e. some membrane interaction & both cells retaining their original phenotypes), homodimeric (i.e. both cells with fused membranes & sharing cytoplasm), cannibalistic (i.e. CTC within a CD14+ macrophage & cells retaining their individual phenotypes), binucleated (i.e. both cells completely merge & becoming one cell with dual expression phenotypes), and hyperploidy (i.e. multiple cells merge to form a large polyploid cell). As CTCs & TMFs are isolated in conjunction from a single blood sample, we evaluated both CTCs & all TMF subtypes to determine their prognostic and predictive values for aggressiveness of disease. Methods: We categorized and enumerated the 5 forms of TMFs from a prospective pilot study using n=120 mBC pts that were starting new lines of treatment. Whole peripheral blood (7.5mL) was procured, filtered and stained using cytokeratin & CD45/CD14 to identify CTCs & TMFs. We compared the presence of the various types of TMFs & CTCs to pt’s progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs), analyzed by censored univariate analysis based on RECIST v1.1 over 24 months. Results: CTCs were found in 39% (n=47/120), with partial fusion TMFs in 26% (n=31/120), homodimeric 2% (n=2/120), cannibalistic 0% (n=0/120), binucleated 2% (n=2/120), hyperploidy 93% (n=113/120). Presence of CTCs alone, binucleated, or hyperploidy TMFs were not prognostic for PFS (p=0.0988, p=0.4120, & p=0.7615 respectively), or OS (p=0.2015, p=0.0845, p=0.1121 respectively). However, TMFs with partial or homodimeric fusion were prognostic for worse PFS (HR=2.2, p=0.0063 and HR=6.0, p=0.0305, respectively) and for worse OS (HR=2.8, p=0.0037 and HR=8.8, p=0.0271, respectively). Furthermore, combining pts with any TMFs into one group, minus hyperploidy, was highly significant for worse PFS (HR=2.9, 95%CI 1.7-5.0, p=0.0002) and worse OS (HR=3.8, 95%CI 2.0-7.5, p=0.0002), though certain subtypes of hyperploidy were also indicative of worse outcomes. Conclusion: The study of TMFs is relativity limited and their existence is new in oncology. Here we detected and described TMFs in the blood of mBC pts while demonstrating an association with poor clinical outcomes. These data suggest TMF involvement in the pathogenic cascade of cancer and further understanding of their biology may be important in the study of tumorigenesis. Citation Format: Daniel L. Adams, R. Katherine Alpaugh, Rena G. Lapidus, Saranya Chumsri, Carolina Reduzzi, Kirby P. Gardner, Cha-Mei Tang, Giuseppe Del Priore, William V. Williams, Massimo Cristofanilli. Tumor macrophage fusion cells detected in the circulation of metastatic breast cancer patients is prognostic for rapid progression and death [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 2310.

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.

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.