Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient’s overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

The Blood Profiling Atlas in Cancer (BLOODPAC) Consortium is a collaborative effort involving stakeholders from the public, industry, academia, and regulatory agencies focused on developing shared best practices on liquid biopsy. This report describes the results from the JFDI (Just Freaking Do It) study, a BLOODPAC initiative to develop standards on the use of contrived materials mimicking cell-free circulating tumor DNA, to comparatively evaluate clinical laboratory testing procedures. Nine independent laboratories tested the concordance, sensitivity, and specificity of commercially available contrived materials with known variant-allele frequencies (VAFs) ranging from 0.1% to 5.0%. Each participating laboratory utilized its own proprietary evaluation procedures. The results demonstrated high levels of concordance and sensitivity at VAFs of >0.1%, but reduced concordance and sensitivity at a VAF of 0.1%; these findings were similar to those from previous studies, suggesting that commercially available contrived materials can support the evaluation of testing procedures across multiple technologies. Such materials may enable more objective comparisons of results on materials formulated in-house at each center in multicenter trials. A unique goal of the collaborative effort was to develop a data resource, the BLOODPAC Data Commons, now available to the liquid-biopsy community for further study. This resource can be used to support independent evaluations of results, data extension through data integration and new studies, and retrospective evaluation of data collection.

Background: Heterogeneous circulating tumor cells (CTCs) have been implicated in the formation of new metastases. However, circulating cells expressing both tumor and immune cell proteins are often dismissed as insignificant findings in CTC studies. Methods: Two non-contemporaneous blood samples from a metastatic breast cancer patient were analyzed using an enrichment-free platform to identify canonical, epithelial-only CTCs (CD45-/cytokeratin + , epi.CTCs) and CD45 + /cytokeratin+ immune-like CTCs (im.CTCs). Single cells from both samples were subjected to copy number and protein expression profiling. A cohort of 36 metastatic breast cancer patients was then analyzed to search for additional cases with im.CTCs. Results: Here, we identified and characterized a population of CTCs exhibiting an immune-like state. In two samples from an index patient, im.CTCs outnumbered epi.CTCs, comprising >97% of the CTC population. Single-cell copy number analysis of 43 im.CTCs and 30 epi.CTCs revealed clonal alterations across both populations, confirming a shared tumor origin. Furthermore, im.CTCs contained pseudo-diploid profiles that did not reflect dilution from the addition of a normal diploid genome, indicating that they were unlikely to have originated from tumor-immune cell fusion. Protein expression analysis showed that im.CTCs express CD45 as well as other immune-related markers, such as CD3 and CD4, and the cancer stemness marker, CD44. Subsequent analysis of a metastatic breast cancer cohort identified an additional patient harboring im.CTCs with the same tumor-derived, non-fusion genome as in the index case. Conclusions: Collectively, these genomic and proteomic features distinguish im.CTCs from previously reported circulating cells may represent a novel form of tumor cell plasticity.

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics.

The chief goal of the Blood Profiling Atlas in Cancer (BloodPAC) consortium is to promote collaborative efforts that support the development and implementation of liquid biopsy tests. Here, we report the results of a pilot study conducted by three BloodPAC members that aimed to demonstrate a multisite liquid biopsy testing framework using longitudinal blood specimens from 38 patients with metastatic breast cancer. Three laboratories receiving identical samples from two clinical sites each applied a different targeted sequencing platform to analyze mutations in cell-free DNA (cfDNA). The resulting mutational profiles reflected common breast cancer alterations, including clinically actionable mutations for 40% of hormone- receptor-positive patients. In 12 genes with shared target regions across sequencing panels, perfect inter-assay concordance was also observed for mutations detected above the lowest common assay limit of detection. Whole-genome copy number profiling of cfDNA and circulating tumor cells (CTCs) further revealed marked heterogeneity in copy number alterations and cfDNA tumor fractions across patients. Additionally, comparison of tumor fraction and CTC abundance demonstrated the complementary nature of cfDNA and CTC analyses. Overall, the framework described in this study may serve as a resource for future trials aiming to identify multimodal liquid biopsy biomarkers to guide clinical care.

Usability is an overlooked aspect of implementing lab-based assays, particularly novel assays in low-resource-settings. Esoteric instructions can lead to irreproducible test results and patient harm. To address these issues, we developed a software application based on “Aquarium”, a laboratory-operating system run on a computer tablet that provides step-by-step digital interactive instructions, protocol management, and sample tracking. Aquarium was paired with a near point-of-care HIV drug resistance test, “OLA-Simple”, that detects mutations associated with virologic failure. In this observational study we evaluated the performance of Aquarium in guiding untrained users through the multi-step laboratory protocol with little supervision. To evaluate the training by Aquarium software we conducted a feasibility study in a laboratory at Coptic Hope Center in Nairobi, Kenya. Twelve volunteers who were unfamiliar with the kit performed the test on blinded samples (2 blood specimens; 5 codons/sample). Steps guided by Aquarium included: CD4+ T-Cell separation, PCR, ligation, detection, and interpretation of test results. Participants filled out a short survey regarding their demographics and experience with the software and kit. None of the laboratory technicians had prior experience performing CD4+ separation and 7/12 had no experience performing laboratory-based molecular assays. 12/12 isolated CD4+ T cells from whole blood with yields comparable to isolations performed by trained personnel. The OLA-Simple workflow was completed by all, with genotyping results interpreted correctly by unaided-eye in 108/120 (90%) and by software in 116/120 (97%) of codons analyzed. In the surveys, participants favorably assessed the use of software guidance. The Aquarium digital instructions enabled first-time users in Kenya to complete the OLA-simple kit workflow with minimal training. Aquarium could increase the accessibility of laboratory assays in low-resource-settings and potentially standardize implementation of clinical laboratory tests.