Creatv MicroTech (United States)

e16464 Background: Pancreatic cancer (PC) is a highly aggressive cancer accounting for ~7% of all cancer related deaths in the United States. In PC, Carbohydrate antigen 19-9 (CA19-9) is blood-based protein biomarker which linearly correlates with disease stage and at ≥37 U/mL is associated with worse clinical outcomes, though its clinical utilization is limited in ~20% PC patients (pts) with low/no CA19-9 secreting tumors. Recently, a phagocytic tumor macrophage found in the blood in parallel with CA19-9 (e.g. cancer-associated macrophage-like cells [CAMLs]), appears to be independently associated with poorer prognosis in PC, when engorged ≥50 µm. However, the relationship between CAMLs & CA19-9 remains unexplored. In this pilot study, we recruited n = 68 PC pts with clinically resectable or borderline resectable PC, to assess ≥50 µm CAMLs & ≥37 U/mL CA19-9 against progression free survival (PFS) and overall survival (OS) over 5 years. Methods: We initiated a prospective pilot study to procure blood samples from pts (n = 68) referred for surgical resection with clinically staged resectable (n = 50) or borderline resectable (n = 18) PC. Anonymized pre-surgical blood samples (7.5mL) were processed via CellSieve microfiltration, isolated, and enumerated for number of CAMLs ≥50 µm. In parallel, CA19-9 levels ≥37 U/mL were measured. The association of CA19-9 & CAMLs against PFS & OS was analyzed at 60 months, with hazard ratios (HRs) calculated by censored univariate and multivariate analyses. Results: CA19-9 was available for 96% (n = 65/68) of pts and CAMLs available for 84% (n = 57/68), with (n = 4) missing a pre-surgical blood draw, (n = 5) failing the CAML assay, and (n = 2) receiving treatment offsite. 55 pts had both CA19-9 and CAML results available. 48% (n = 31/65) of pts had CA19-9 ≥37 U/mL which was not significant for PFS (HR = 1.6, 95%CI = 0.8-2.9, p = 0.2230), but was significant for worse OS (HR = 2.0, 95%CI = 1.0-3.8, p = 0.0486). 68% (n = 39/57) of pts had CAMLs ≥50 µm which was significant for worse PFS (HR = 3.5, 95%CI = 1.8-6.7, p = 0.0004) and OS (HR = 3.7, 95%CI = 1.9-7.3, p = 0.0003). 40% (n = 22/55) of pts had both CA19-9 ≥37 U/mL & CAMLs ≥50 µm which was significant for worse PFS (HR = 5.0, 95%CI = 2.2-11.3, p = 0.0002) & OS (HR = 7.9, 95%CI = 3.4-18.2, p < 0.0001). At post-surgical pathological assessment it was found that the percentage of pts with locally advanced (LA) or metastatic PC (mPC) disease was, 86% when CAMLs ≥50 µm & CA19-9 ≥37 U/mL, 71% when CAMLs ≥50 µm but CA19-9 < 37 U/mL, 25% when CAMLs < 50 µm but CA19-9 ≥37 U/mL, or 25% when CAMLs < 50 µm & CA19-9 < 37 U/mL. Conclusions: The combination of CAMLs & CA19-9 shows promise as a non-invasive biomarker for stratifying pts with worse clinical outcomes and possibly identifying resectable pts with more advanced PC. Given these results, future studies with larger cohorts should evaluate and refine any potential use in guiding treatment decisions in PC.

Abstract Studies have described the efficacy of immunotherapies (IMT) utilizing programmed death 1 receptor and its ligand (PD-L1) for treating solid tumors. However, many patients (pts) fail to respond to IMT, underscoring the need for better predictive biomarkers for improved pt identification and stratification. Poor IMT responses are often attributed to the dynamic nature of PD-L1 which may change after chemotherapy or radiation and is quantified by static immunostaining. Recently, studies have described PD-L1 upregulation in giant phagocytic stromal cells, i.e. Cancer associated macrophage-like cells (CAML), circulating macrophages that have engulfed tumors before entering circulation and may predict IMT responses. We conducted a pilot study to monitor the peripheral blood of n=46 metastatic cancer pts undergoing systemic treatment with IMT in combination with other therapies, to evaluate CAML PD-L1 prior to and post IMT induction with clinical correlation at 2 years. In a prospective pilot study of n=46 metastatic cancer pts, breast (n=14), non-small cell lung (n=24), and renal cell (n=8), all starting new lines of systemic chemotherapy in combination with IMT (pembrolizumab [n=30], nivolumab [n=13], Durva [n=1], or atezolizumab [n=3]) for newly diagnosed recurrent metastasis (n=24) or with previously treated progressive metastatic disease (n=22). We isolated CAMLs from 7.5 ml baseline (T0) blood using the LifeTracDx® PD-L1 test and scored PD-L1 as high or low. If possible, a follow-up sample (T1) was taken (∼48 days) after IMT induction. Pts’ progressive free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate analysis based on RECIST v1.1 over 2 years. T0 PD-L1 CAML data was available for 89% (n=41/46) of pts, with 39% (n=16/41) having high CAML PD-L1 which was not correlated with improved PFS (HR=0.9, p=0.985, CI=0.5-1.9) or OS (HR=1.0, p=0.899, CI=0.4-2.2). T1 PD-L1 CAML data was available for 74% (n=34/46) of pts, with 53% (n=18/34) having high CAML PD-L1, which significantly correlated with improved PFS (HR=2.9, p=0.012, CI=1.3-6.4) and OS (HR=5.5, p=0.0005, CI=2.2-13.6). In comparing CAML PD-L1 change post IMT, it was found that consistently low PD-L1 at T0 & T1 had the poorest responses, median PFS (mPFS)=2.3 months and median OS (mOS)=5.5 months. In contrast, consistently high PD-L1 at T0 & T1 had better responses, mPFS=6.1 months and mOS=16.7 months. Further, pts who increased in CAML PD-L1 after IMT had the best response rates, mPFS=10.3 months and mOS=14.8 months. Here, we describe a cancer agnostic blood-based biopsy to monitor dynamic PD-L1 changes in circulating tumor immune cells and predict clinical benefit to PD-L1 IMTs in several cancer types. While this initial pilot study appears to stratify pts with better IMT responses, larger validation studies are needed. Citation Format: Dimpal M. Kasabwala, Steven H. Lin, Massimo Cristofanilli, Carolina Reduzzi, Cha-Mei Tang, Thai H. Ho H. Ho, Daniel L. Adams. Monitoring PD-L1 expression in cancer associated macrophage-like cells in blood predicts clinical outcomes in metastatic cancer patients treated with PD-L1 immunotherapies [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 2061.

A diverse group of professionals gathered in Brasília for the VI Brazilian Symposium on Toxoplasmosis and III International Symposium on Toxoplasmosis from October 17 to 19, 2023. Guided by the ‘One Health’ concept, the event addressed toxoplasmosis in its human, animal, and environmental dimensions. Participants discussed strategies to improve the prevention, diagnosis, and treatment of the disease in Brazil, also emphasizing a Pan-American cooperation. The resulting document, the Brasília Letter: Proposal for Toxoplasmosis Control in Brazil with a Multidisciplinary Approach and a Pan-American Cooperation Perspective, synthesizes the specialists’ concerns and proposes actions for the development of public health policies in Brazil and other countries in the Americas. This initiative aims to promote significant advances in toxoplasmosis control, benefiting society’s health on a continentaland global scale.

Abstract Background: Metastatic Prostate cancer (mPCa) mortality rates are high despite numerous treatment options against various mechanisms of action, including targeted therapies, i.e. prostate specific membrane antigen (PSMA)-targeted therapy (Pluvicto) or anti-androgen receptor (AR) (Enzulamide, Biculamide). Further, tumor biomarkers (i.e. PSMA and AR) can change over time and with resistance mechanisms that develop after treatments. This suggests that primary biopsies may not represent later PCa tumors necessitating a way to identify patients that may respond to later line therapies. Blood based biopsies can be used to monitor PCa treatment, which can include circulating tumor cells (CTCs) and the newly discovered cancer associated macrophage like cells (CAMLs), a cancer specific circulating phagocytic stromal cells. Interestingly, while PSMA & AR have been identified in CTCs, they have not been evaluated in CAMLs, nor have PSMA & AR expressions in CTCs & CAMLs been evaluated in anti-AR therapy settings. We measured CAMLs & CTCs in mPCa to evaluate their PSMA & AR expression, including in patients treated with anti-AR therapy. Methods: We evaluated CAMLs & CTCs in a multi-institutional prospective pilot study using n=30 mPCa patients with progressive disease, prior to starting a new line of therapy (T0). Whole peripheral blood (7.5mL) was filtered for CAMLs & CTCs and stained for PSMA (n=15) & AR (n=15). In addition, 15 patients were treated with anti-AR therapy as standard of care and responses by PET/CT were compared against AR expression. When possible, follow up samples (T1) were also procured. Results: CTCs were identified in 16% (n=5/30) of patients and CAMLs were identified in 96% (n=29/30), with 53% of patients having hyperengorged CAMLs ≥50 μm (n=16/30). CTCs in patients were not statistically significant for worse outcomes PFS HR= 0.1748, p=0.546 nor OS (HR= 1.303, p=0.865), nor were CAMLs prognostic for PFS or OS. However, patients with ≥50 μm CAMLs did have significantly worse PFS (HR= 5.9, CI95% 2.2-16.3, p=0.001) and worse OS (HR= 3.1, CI95% 1.3-7.1, p=0.016). Further, PSMA CAML/CTC expression was high in 60% (n=9/15) of patients and AR CAML/CTC expression was high in 53% (n=8/15). Of the n=15 patients who received anti-AR therapy after T0, high AR expressing CAMLs/CTCs (n=8/15) had a ~250% better mPFS of 10.3 months (mons) (progressing at 2.2-32.6 mons) vs low AR CAMLs/CTCs mPFS of 4 mons (progressing at 0.2-7.2 mons). Additional mOS analysis from T1 samples and 2-year survival is ongoing. Conclusion: We utilized liquid biopsies to monitor the expression of two common cell surface receptors (PSMA & AR) in CAMLs and CTCs in mPCa. Further, in a small subset of patients treated with AR therapy, high AR CTC/CAML expression appeared to correlate with better response rates, though larger prospective studies are needed. Citation Format: Dimpal M. Kasabwala, Raymond C. Bergan, R. Katherine Alpaugh, Daniel C. Danila, Tuan L. Chuang, Brenda Y. Hurtado, Daniel L. Adams. Monitoring prostate specific membrane antigen or androgen receptor expression on circulating stromal cells in advanced prostate cancer patients and their correlation with patient response [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 3690.

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.

High-resolution, x-ray phase contrast microscopy, a key technique with promising potential in biomedical imaging and diagnostics, is based on narrow-slit high-aspect-ratio gold gratings. We present the development, fabrication details, and experimental testing of the freestanding 10 μm thick gold membrane masks with an array of 0.9–1.5 μm void slit apertures for a novel low-energy x-ray microscope. The overall mask size is 4 mm × 4 mm, with a grating pitch of 7.5 μm, 6.0–6.6 μm wide gold bars are supported by 3 μm wide crosslinks at 400 μm intervals. The fabrication process is based on gold electroplating into a silicon mold coated with various thin films to form a voltage barrier, plating base, and sacrificial layer, followed by the mold removal to obtain the freestanding gold membrane with void slit apertures. We discuss key aspects for the materials and processes, including gold structures homogeneity, residual stresses, and prevention of collapsing of the grid elements. We further demonstrate the possibility to obtain high-resolution, high contrast 2D images of biological samples using an incoherent, rotating anode x-ray tube.

Abstract Background: SV-BR-1-GM is a GM-CSF-engineered breast cancer cell line employed – after irradiation – as a targeted immunotherapy for advanced breast cancer. Tumor regressions at metastatic sites have been observed, most notably in patients with HLA allele matches to the cell line. We are assessing SV-BR-1-GM in the phase IIa portion of a phase I/IIa clinical trial in metastatic and locally recurrent breast cancer (ClinicalTrials.gov identifier NCT03066947). Additionally, we are co-developing a companion diagnostic (BriaDX™) to identify patients likely to respond to SV-BR-1-GM. Currently, BriaDX™ consists of HLA typing; however, we have begun assessing biomarkers in sera, lymphocyte characteristics, circulating tumor cells (CTCs), and cancer-associated macrophage-like cells (CAMLs) from patients collected at baseline and after inoculation of SV-BR-1-GM to layer in additional components to improve accuracy, with the number/subtyping of CTCs and CAMLs being prognostic indicators. Methods: Subjects are pretreated with low-dose cyclophosphamide to reduce immune suppression. SV-BR-1-GM is inoculated intradermally with follow-up local injections of IFNα2. Cycles are every 2 weeks x 3, then monthly. HLA typing was conducted via LabType R-SSO Kits (One Lambda). Cytokines were measured via single- or multiplex assays. Anti-SV-BR-1 antibodies were determined by incubation of SV-BR-1 cells with diluted patient sera followed by staining with a fluorescently-labeled anti-IgG antibody and detection by flow cytometry. CTCs and CAMLs were evaluated by CellSieve™ at Creatv MicroTech. Results: To date, 16 clinical trial subjects have been inoculated with the SV-BR-1-GM regimen as rescue immunotherapy. All were treatment refractory and had received a median of 4.5 prior chemo/biologic therapy regimens (range 1-13). Two of the 16 patients remained on study for ≥3 months (5 cycles) with 4 patients currently on study not having reached the 3-month evaluation time point. Objective regression of tumor was seen in 2 subjects. One subject had virtually complete regression of 20 of 20 lung metastases noted at 3 and 6 months (but with progressive bone and liver metastases). Another subject had improvement of chest wall metastases and quality of life but expired due to nontreatment-related causes. Response appeared to correlate with HLA allele-matching to SV-BR-1-GM. Anti-SV-BR-1 antibody titers increased in several patients. Among the cytokines assessed, interleukin (IL)-8 levels increased in HLA-DRB3 allele-matched subjects after SV-BR-1-GM inoculation. Of 15 patients evaluated, CTCs were present in 6 patients at baseline while CAMLs were present in all 15. Five of 5 patients evaluated for PD-L1 expression had mostly low-to-medium expression of PD-L1 on their CTCs/CAMLs. In the patient who had regression of lung metastases but progression of liver metastases, PD-L1 expression and maximum CAML size increased, but the number of CAMLs decreased during treatment. CAML number also decreased in a patient who reached the 3-month evaluation visit without progression and in a patient with inflammatory breast cancer who dropped out due to worsening inflammation. Conclusions: In addition to the patients’ HLA types, several pharmacodynamic parameters correlated with tumor regression and/or HLA matching status. CTCs or CAMLs are frequently detectable in this population, and PD-L1 expression appears common on these cells. Both CAML number and size appear to correlate with response, though larger studies are needed. Future steps include evaluation of SV-BR-1-GM with checkpoint inhibitors. Citation Format: Markus D. Lacher, Sanne Graeve, Vivekananda (Vivek) Sunkari, Daniel L. Adams, Cha-Mei Tang, Pete Amstutz, Charles L. Wiseman, George E. Peoples, William V. Williams. SV-BR-1-GM, a whole-cell targeted immunotherapy for advanced breast cancer: Pharmacodynamic markers of response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A012.

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