Aldakkak, Mohammed

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.

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.

e16260 Background: Therapeutic inhibition of constitutive signaling mediated by mutated KRAS in PDAC remains a challenge except for modest success reported with KRAS G12C inhibition. A combinatorial strategy utilizing simultaneous MEK and autophagy inhibition holds therapeutic promise based on mechanism of action and preclinical data. We described characteristics and outcomes of patients (pts) treated with MEK-inh and HCQ at our institution. Methods: Ten KRAS-mutated advanced PDAC pts were treated with trametinib-HCQ (n = 9) or cobimetinib-HCQ (n = 1) off label due to lack of standard treatment options or toxicity concerns with cytotoxic systemic therapy. Trametinib dose was 2 mg once daily orally, Cobimetinib dose was 20 mg BID orally for 3/4 weeks cycles. HCQ was started at 200 mg BID and up-titrated weekly to 600 mg BID. Description of baseline and treatment (tx) characteristics, safety and efficacy is provided. Results: Median age at diagnosis was 61.3 years, and 7 pts were female. The number of prior lines of tx were 0/1/2/3/4 in 3/2/1/2/2 pts, respectively. KRAS mutations were: G12R/G12D/G12V/Q61H in 6/2/1/1 pts. Median overall survival was 6.6 months (m) in all pts, and 6.6/1.7 m in KRAS G12R/other KRAS (p = 0.31). Median progression-free survival was 5.7/6.2/1.5 m in all/ KRAS G12R/other KRAS (p = 0.16). Among 8 pts with evaluable response, 1 (12%) had partial response ( KRAS G12R) and 4 (50%) stable disease (3/4 KRAS G12R) as best response with disease control rate of 63%/80%/33% in total/ KRAS G12R/other KRAS (p = 0.29). Toxicity data are summarized in table 1. Conclusions: MEK-inh-HCQ demonstrated modest efficacy and manageable toxicities among KRAS G12R PDAC pts. Unlike G12D and G12V mutations in the KRAS gene, G12R is defective of conductive interactions for both PI3Ka and NF1. This ultimately results in a weakened signal being shunted through MAPK cascade and provides a unique opportunity where MEK inh can ablate signaling without the alternate pathways and WT-RAS isoforms compensating. Furthermore, as activation of PI3Ka is known to suppress autophagy, its lack of activation by KRAS G12R further sensitizes cells to HCQ. The combination therapy MEK-inh-HCQ is therefore mechanistically-rationale and warrants the further investigation of KRAS G12R as an actionable biomarker.[Table: see text]