Tsai, Susan

Abstract Background: In metastatic cancer, Circulating Tumor cells (CTCs) are established prognostic indictors of patients (pts) less likely to respond to new lines of systemic therapy, with poor clinical outcomes, such as shorter progression free survival (PFS) and overall survival (OS). However, CTCs are typically found in specific malignancies (breast, prostate & colon), often in <20% of pts with metastatic disease, and pts without CTCs may also rapidly progress. Recently, an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in >90% of metastatic cancer pts, and whose phagocytic engorgement appears to correlate with poor outcomes, independent of CTCs. As CTCs and CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for outcomes, we evaluated their utilization prior to induction of new systemic therapy in 6 types of metastatic cancer to model pt risk stratification based on 2 year outcomes. Methods: A prospective 2 year blind multi-institutional study was undertaken to model CTCs and CAMLs in prognosticating outcomes prior to induction of a new line of systemic therapy (n=233) in metastatic: Breast (n=60), Prostate (n=40), Pancreas (n=25), Colon (n=28), Renal Cell Carcinoma (RCC) (n=39), and Lung (n=40). Blood was filtered by CellSieveTM filters with subtypes of CTCs & hyper-enlarged CAMLs (≥100µm) enumerated. A machine learning algorithm was trained on this initial data set to develop predictive models which could stratify pt populations by risk for likelihood of PFS & OS over 2 years, including known clinical variables. Results: CTCs were absent in 80% (n=185/233) of pts, and their absence was prognostic for better PFS (HR=1.6, p=0.046), but not OS (HR=1.3, p=0.2045). In parallel, enlarged CAMLs (≥100µm) were found in 28% (n=51/185) of pts without CTCs, and were also prognostic for worse PFS (HR=2.0, p=0.0082) and OS (HR=1.9, p=0.0412). Specifically, CTCs were found in 55% (n=33) breast, 20% (n=8) prostate, 12% (n=3) pancreas, 11% (n=3) colon, 0% (n=0) RCC, and 0% (n=0) lung pts. Enlarged CAMLs were found in 45% (n=27) breast, 20% (n=8) prostate, 44% (n=11) pancreas, 44% (n=17) colon, 18% (n=7) RCC and 20% (n=8) lung pts. Overall, models indicated that ≥1 CTC (n=47) had mPFS=3.9 & mOS=14.9, while pts with 0 CTCs and ≥100µm CAMLs (n=51) had a mPFS=6.9 & mOS=13.8, and pts with 0 CTCs and <100µm CAMLs (n=134) had a mPFS=10.7 & mOS>24 months. Conclusions: These initial models confirm that both CTCs and enlarged CAMLs are prognostic indicators of worse PFS & OS. The simultaneous quantification both CTCs and CAMLs allows for more accurate pan-cancer risk stratification in an array of cancer pt populations. Additional clinical variables incorporated into the models may allow better risk subtyping and possibly forecast optimal treatment regimens. Citation Format: Daniel L. Adams, Steven H. Lin, Massimo Cristofanilli, Carolina Reduzzi, Saranya Chumsri, Martin J. Edelman, Susan Tsai, Raymond C. Bergan, Mohammed Aldakkak, Thai H. Ho, Cha-Mei Tang. Combining circulating tumor cells and cancer associated macrophage-like cells enhances risk stratification models in pan-cancer metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1068.

Abstract Circulating tumor cells (CTCs) are cancer cells that detach from tumors, enter the blood and seed distant sites. CTCs spread as single cells, or cluster aggregates, and can be heterogenous with differing biological phenotypes, i.e. epithelial, mesenchymal, progenitor, & stem cell. Three clinically relevant CTC subtypes are often described in patients (pts) 1) CTCs with normal epithelial traits (strong filamentous cytokeratin (CK+ & CD45-/CD14-), 2) Epithelial-mesenchymal CTCs (EMTs), cancer cells with downregulated epithelial & upregulated mesenchymal traits (low diffused CK+, CD45-/CD14-) and 3) tumor-macrophage fusion cells (TMFCs), cancer cells that fuse with macrophages (polynucleated DAPI+, diffuse CK+ and CD14+/CD45+). To better elucidate the biological and clinical importance of the 3 CTC subtypes, we isolated CTCs by a marker agnostic method from the blood of n=49 metastatic cancer pts. We then enumerated and phenotypically subtyped CTCs comparing clinical utility of clustering and CTC heterogeneity over 2 years. Peripheral blood samples were collected from 49 metastatic cancer pts, breast (n=38), pancreatic (n=9), colon (n=3), lung (n=1), and prostate (n=1). Whole blood (7.5mL) was filtered by size exclusion using CellSieve microfilters and fluorescently stained for CD45, CD14, CK, and DAPI to identify CTCs, EMTs and TMFCs. Cells were imaged, enumerated and fluors quenched by the QUAS-R technique; followed by re-stained for CD44, CD24, CD133, CD144, N-Cadherin, EpCAM, and Vimentin. Markers were categorized as negative, low, medium, or high. A heterogeneity biomarker score was developed as high, 2-3 log deviations between marker expression, or low 0-1. Pt progressive free survival (PFS) and overall survival (OS) were analyzed over 2 years. CTCs were found in 40% of pts, EMTs in 51%, & TMFCs in ∼93%. For subtyping, CD44 was in CTCs 88%, EMTs 59%, & TMFCs 91%; CD24 in CTCs 40%, EMTs 41%, & TMFCs 6%; CD133 in CTCs 100%, EMTs 8%, & TMFCs 100%; CD144 in CTCs 100%, EMTs 77%, & TMFCs 82%; N-cadherin in CTCs 100%, EMTs 0%, & TMFCs 45%; Vimentin in CTCs 51%, EMTs 100%, & TMFCs 83%; EpCAM in CTCs 74%, EMTs 12%, & TMFCs 20%. CTCs and EMTs were found to have high heterogeneity in 30% of samples in both individual cells and clusters, with a high biomarker heterogeneity significantly correlated with poorer outcomes (PFS: HR=15.4, p=0.0014, CI=3.4-68.9; OS: HR=12.4, p=0.0056, CI=2.6-59.1). TMFCs were found to have high heterogeneity, 40% of samples, but this was not correlated with outcomes. In this pilot study, it appeared that isolated CTC, EMTs and TMFCs subtypes have differing heterogenous expressions of epithelial, mesenchymal, progenitor, and stem cell markers. Further, high rates biomarker heterogeneity in CTCs and EMTs, but not TMFCs, correlated with faster rates of progression & death. Larger validation experiments with biomarker stratification is ongoing. Citation Format: Alexis B. Duffy, Massimo Cristofanilli, Carolina Reduzzi, Cha-Mei Tang, Saranya Chumsri, Susan Tsai, Mohammed Aldakkak, Daniel L. Adams. Heterogeneity of epithelial, mesenchymal, progenitor, and stem cell phenotypes on CTCs correlates to clinical outcomes in metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1989.

Abstract Tumor associated macrophages are known to fuse with cancer cells resulting in the formation of a cellular phenomenon called tumor-macrophage fusion cells (TMFCs). TMFCs are a highly aggressive cell subtype that may move into circulation, evade immune surveillance, and possibly aid in seeding of metastatic sites. The formation of TMFCs involves CD47, a ubiquitous surface receptor that acts as a “don’t eat me” signal, interacting with the macrophage protein SIRPa, a down regulator of phagocytosis, and syncytin-1, a mediator of hyperploidy cancer fusion. In this analysis, we isolated circulating TMFCs from the blood of n=39 metastatic cancer patients (pts) to evaluate the expressions of CD47, SIRPa and syncytin-1 as it relates to stable or progressive disease via 30 day imaging by PET scan. We conducted a prospective pilot study of n=39 pts with pathologically confirmed metastatic breast (n=17), prostate (n=7), pancreas (n=7), or colon (n=8) cancer receiving standard of care systemic therapy. We isolated TMFCs from 7.5ml peripheral blood using a CellSieve microfilter, and stained with CD47, SIRPa, & syncytin-1. TMFCs were identified based on large cell size (>30 µm) & multinucleated structure, and were categorized into 4 distinct subtypes: A) >2 distinct non-contacting nuclei within a TMFC, B) 2 distinct non-contacting nuclei, C) 2 contacting nuclei, D) a single multinucleated nuclei. CD47, SIRPa, and syncytin-1 expressions were categorized as negative, low, or high. TMFCs subtypes and marker expressions were compared to pts’ clinical status (i.e. progressive, stable or responding) at first available PET/CT scans after blood draw (∼45 days). TMFCs were identified in 72% of pts (n=28/39), average 8 cells/sample, and ranged 1-77 cells. Of the TMFCs, 12% were subtype A, 11% as B, 11% as C, and 66% as D. CD47 expression was seen in 100% of TMFs, with low expression in 39% of subtype A cells, 58% subtype B, 39% subtype C, & 62% subtype D. Notably, CD47 was significantly lower (p=0.010) in subtype B than other groups. Further, the expression of syncytin-1 in subtype B cells was significantly lower (p=0.02) than in other subtypes, and subtype D cells had 75% higher syncytin-1 localized to the nuclear regions. No correlations were found in the SIRPa expression and subtypes. Interestingly, progressing pts had 200% more TMFCs, more subtype B (83%), and significantly lower (p<0.001) CD47 expressing TMFCs than non-progressing pts. In this pilot study on the expression profiles of circulating TMFCs, it appears that specific subtypes of TMFC with low CD47 expression may be associated with progressive disease. Interestingly, this contrasts existing studies on primary tumors which often correlate higher CD47 with more aggressive cancers. This finding suggests that low CD47 cancer cells circulate blood in some pts with progressing disease and may be pertinent to efforts targeting CD47 in cancer. Citation Format: Sonia Muthuraj, Massimo Cristofanilli, Saranya Chumsri, Susan Tsai, Raymond C. Bergan, Cha-Mei Tang, Daniel L. Adams. Low CD47 expression in specific populations of tumor macrophage fusion cells in blood correlates with progressive disease in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1979.

Importance Neoadjuvant therapy (NT) is an increasingly used treatment strategy for patients with localized pancreatic ductal adenocarcinoma (PDAC). Little research has been conducted on cancer care delivery during NT, and the standards for optimal delivery of NT have not been defined. Objective To develop consensus best practices for delivering NT to patients with localized PDAC. Design, Setting, and Participants This study used a modified Delphi approach consisting of 2 rounds of voting, and a series of virtual conferences (from October to December 2023) to reach expert consensus on candidate best practice statements generated from a systematic review of the literature and expert opinion. An interdisciplinary panel was formed including 47 North American experts from surgical, medical, and radiation oncology, radiology, pathology, gastroenterology, integrative oncology, anesthesia, pharmacy, nursing, cancer care delivery research, and nutrition as well as patient and caregiver stakeholders. Main Outcome and Measures Statements that reached 75% agreement or greater were included in final consensus statements. Results Of the 47 participating panel members, 27 (57.64%) were male, and the mean (SD) age was 47.6 (8.2) years. Physicians reported completing training a mean (SD) 14.6 (8.6) years prior and seeing a mean (SD) 110.6 (38.4) patients with PDAC annually; 35 (77.7%) were in academic practice. Final consensus was reached on 82 best practices for delivering NT. Of these, 38 statements focused on pre-NT practices, including diagnosis and staging (n = 15), evaluation and optimization (n = 20), and decision-making (n = 3); 29 statements defined best practices during NT, including initiation (n = 3), delivery of therapy (n = 8), restaging practices (n = 12), and management of complications during NT (n = 6); and 15 best practices were identified to guide treatment post-NT, focusing on surgery (n = 7), pathology (n = 4), and follow-up (n = 3). Conclusions Using a modified Delphi consensus technique, best practice guidelines were developed focusing on the optimal standards for delivering NT to patients with localized PDAC. Given the prognostic importance of completing multimodality therapy, efforts to standardize and optimize the delivery of NT represent an immediate opportunity to decrease care variation and improve outcomes for patients with PDAC. Future research should focus on validating and implementing best practice standards into clinical practice.

3053 Background: Recently, early studies have identified circulating stromal cells (CStCs), i.e. Cancer Associated Macrophage-Like cells (CAMLs), in patients (pts) with newly diagnosed invasive cancers, which are phagocytic myeloid immune cells emanating from primary tumors. However, no study has evaluated CAMLs as a pan solid tumor screening tool nor their clinical impact on pt outcomes. We analyzed the blood of untreated newly diagnosed cancer pts (n=441), or in non-malignant conditions (i.e. pancreatic cysts, n=77), or healthy controls (n=100). We found that CAMLs are prevalent (82%) in cancer pt blood, absent in healthy controls (0%) and less common in non-malignant conditions (36%), with correlations to worse clinical outcomes. Methods: Anonymized peripheral blood was taken from n=441 pts before or after confirmation of invasive malignancy [stage I (n=105), stage II (n=125), stage III (n=114), stage IV (n=97) with pathologically confirmed lung (n=84), pancreas (n=61), breast (n=73), prostate (n=65), esophageal (n=34), Renal Cell (n=29), Sarcoma (n=27), or Other (n=68). In addition, anonymized blood was taken from pts with untreated non-malignant conditions including benign breast masses (n=19), Lupus (n=11), liver Cirrhosis (n=6), rising PSA (n=24), or other benign mass (n=17); and from healthy control volunteers (n=100). CAMLs were isolated by CellSieve microfiltration, defined as enlarged polyploid cells with cytokeratin+ and/or CD45/CD14+. Pts were monitored for 0-8 years (median=2.6) for progression free survival (PFS) and overall survival (OS). Results: CAMLs were found in 82% of all cancer pts averaging 5.4 CAMLs, specifically, in 66% of Stage I, 85% Stage II, 84% Stage III, and 92% Stage IV. No CAMLs were found in any healthy controls, but were in 36% of non-malignant pts. CAML sensitivity in cancer vs healthy was 82% (CI95% 78-85%), specificity=100% (CI95% 96-100%), PPV=100% (CI95% 99-100%), NPV=55% (CI95% 50-60%). CAML sensitivity in cancer vs benign was 82% (CI95% 78-85%), specificity=64% (CI95% 32-44%), PPV=93% (CI95% 90-95%), NPV=38% (CI95% 32-44%). CAML presence was significantly associated with worse PFS (HR=2.0, 95%CI 1.4-2.9, p<0.0001) & OS (HR=2.2, 95%CI 1.4-3.3, p=0.0004), with engorged phagocytic CAMLs (>50um), highly significant for worse PFS (HR=3.3, 95%CI 2.5-4.5, p<0.0001) & OS (HR=3.0, 95%CI 2.1-4.3, p<0.0001). Further, in benign pts positive for CAMLs, 2 pts were in situ, 16 had histologies with increased cancer risk (i.e. IPMN, atypical hyperplasia), and 5 pts died within 3 years. Conclusions: CAMLs appear to be a highly sensitive blood based biomarker that can identify invasive carcinoma in all stages of cancer regardless of subtype, but are not found in healthy controls & rare in non-malignant conditions. Further, enlarged phagocytic CAMLs appeared to correlate with shorter PFS and OS prior to treatment induction.