New inflammatory indicators for cell-based liquid biopsy: association of the circulating CD44+/CD24- non-hematopoietic rare cell phenotype with breast cancer residual disease.

BACKGROUND: Breast cancer residual disease assessment in early-stage patients has been challenging and lacks routine identification of adjuvant therapy benefit and objective measure of therapy success. Liquid biopsy assays targeting tumor-derived entities are investigated for minimal residual disease detection, yet perform low in clinical sensitivity. We propose the detection of CD44-related systemic inflammation for the assessment of residual cancer. METHODS: Circulating CD44+/CD45- rare cells from healthy, noncancer- and cancer-afflicted donors were enriched by CD45 depletion and analyzed by immuno-fluorescence microscopy. CD44+ rare cell subtyping was based on cytological feature analysis and referred to as morphological index. AUC analysis was employed for identification of the most cancer-specific CD44+ subtype. RESULTS: The EpCam-/CD44+/CD24-/CD71-/CD45-/DNA+ phenotype alludes to a distinct cell type and was found frequently at concentrations below 5 cells per 5 mL in healthy donors. Marker elevation by at least 5 × on average was observed in all afflicted cohorts. The positive predicted value for the prediction of malignancy-associated systemic inflammation of a CD44+ rare cell subtype with a higher morphological index was 87%. An outlook for the frequency of sustained inflammation in residual cancer may be given to measure 78%. CONCLUSION: The CD44+ rare cell and subtype denotes improvement in detection of residual cancer disease and may provide an objective and alternative measure of disease burden in early-stage breast cancer.

Detection of circulating rare cells benefitted the diagnosis of malignant solitary pulmonary nodules.

INTRODUCTION: Solitary pulmonary nodules (SPNs) are challenging in differentiating between benignancy and malignancy. Therefore, more effective non-invasive biomarkers are urgently needed. The purpose of this investigation was to examine whether circulating rare cells (CRCs) could facilitate the differentiation between benign and malignant SPNs as well as its sensitivity and specificity. METHODS: 164 patients diagnosed with SPNs, 24 healthy volunteers, and 25 patients diagnosed with advanced-stage lung cancer were included. CT/PET-CT images, serum tumor markers, and biopsy results were collected. The CRCs were examined using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) and their relationship with malignant or benign SPNs was analyzed. RESULTS: The total CRC numbers from patients with malignant SPNs diagnosed by biopsy were significantly greater compared to those with benign SPNs (P < 0.0001), but not significantly different from patients with advanced lung cancer (P > 0.05). The total CRCs, with a cut-off value of 21.5 units, showed 67.6% sensitivity and 73.3% specificity [area under curve (AUC) 95% CI, 0.778 (0.666-0.889)] in discriminating benign and malignant SPNs and the triploid CRCs exhibited a high positive likelihood ratio of 8.4, which suggested that CRCs appeared to have a distinct advantage in discriminating benign and malignant SPNs compared to CT/PET-CT images and serum tumor markers and could be a potential screening indicator for lung cancer in the high-risk population. CONCLUSIONS: SE-iFISH could effectively detect CRCs including circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) and the detection of CRCs could benefit the differentiation of patients with benign and malignant SPNs.

Comprehensive Atlas of Circulating Rare Cells Detected by SE-iFISH and Image Scanning Platform in Patients With Various Diseases.

Objective: Circulating rare cells (CRCs) are known as a crucial nucleated cellular response to pathological conditions, yet the landscape of cell types across a wide variety of diseases lacks comprehensive understanding. This study aimed at detecting and presenting a full spectrum of highly heterogeneous CRCs in clinical practice and further explored the characterization of CRC subtypes in distinct biomarker combinations and aneuploid chromosomes among various disease groups. Methods: Peripheral blood was obtained from 2,360 patients with different cancers and non-neoplastic diseases. CRC capture and identification were accomplished using a novel platform integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy with a high-throughput automated image scanning system, on which hemocyte, tumor, epithelial, endothelial, mesenchymal, and stemness biomarkers were immunostained and displayed simultaneously. Double chromosome enumeration probe (CEP8 and CEP12) co-detection was performed on isolated CRCs from an extended trial for two chromosome ploidy patterns. Results: A comprehensive atlas categorizing the diverse CRCs into 71 subtypes outlining was mapped out. The presence of epithelial-mesenchymal transition (EMT) or endothelial-mesenchymal transition (EndoMT), the cells with progenitor property, hematologic CRCs expressing multiple biomarkers, CRCs at "naked nuclei" status, and the rarely reported aneuploid mesenchymal epithelial-endothelial fusion cluster were described. Circulating tumor cells (CTCs) were detected in 2,157 (91.4%) patients; the total numbers of CTCs and circulating tumor-derived endothelial cells (CTECs) were relatively higher in several digestive system cancer types and non-neoplastic infectious diseases (p < 0.05). Co-detection combining CEP8 and CEP12 showed a higher diagnostic specificity on account of 57.27% false negativity of CRC detection through a single probe of CEP8. Conclusions: The alternative biomarkers and chromosomes to be targeted by SE-iFISH and the image scanning platform, along with the comprehensive atlas, offer insight into the heterogeneity of CRCs and reveal potential contributions to specific disease diagnosis and therapeutic target cell discovery.

HGF/c-Met Inhibition as Adjuvant Therapy Improves Outcomes in an Orthotopic Mouse Model of Pancreatic Cancer.

BACKGROUND: Inhibition of hepatocyte growth factor (HGF)/c-MET pathway, a major mediator of pancreatic stellate cell (PSC)-PC cell interactions, retards local and distant cancer progression. This study examines the use of this treatment in preventing PC progression after resection. We further investigate the postulated existence of circulating PSCs (cPSCs) as a mediator of metastatic PC. METHODS: Two orthotopic PC mouse models, produced by implantation of a mixture of luciferase-tagged human pancreatic cancer cells (AsPC-1), and human PSCs were used. Model 1 mice underwent distal pancreatectomy 3-weeks post-implantation (n = 62). One-week post-resection, mice were randomised to four treatments of 8 weeks: (i) IgG, (ii) gemcitabine (G), (iii) HGF/c-MET inhibition (HiCi) and (iv) HiCi + G. Tumour burden was assessed longitudinally by bioluminescence. Circulating tumour cells and cPSCs were enriched by filtration. Tumours of Model 2 mice progressed for 8 weeks prior to the collection of primary tumour, metastases and blood for single-cell RNA-sequencing (scRNA-seq). RESULTS: HiCi treatments: (1) reduced both the risk and rate of disease progression after resection; (2) demonstrated an anti-angiogenic effect on immunohistochemistry; (3) reduced cPSC counts. cPSCs were identified using immunocytochemistry (α-smooth muscle actin+, pan-cytokeratin-, CD45-), and by specific PSC markers. scRNA-seq confirmed the existence of cPSCs and identified potential genes associated with development into cPSCs. CONCLUSIONS: This study is the first to demonstrate the efficacy of adjuvant HGF/c-Met inhibition for PC and provides the first confirmation of the existence of circulating PSCs.

The Blood Circulating Rare Cell Population. What is it and What is it Good For?

Blood contains a diverse cell population of low concentration hematopoietic as well as non-hematopoietic cells. The majority of such rare cells may be bone marrow-derived progenitor and stem cells. This paucity of circulating rare cells, in particular in the peripheral circulation, has led many to believe that bone marrow as well as other organ-related cell egress into the circulation is a response to pathological conditions. Little is known about this, though an increasing body of literature can be found suggesting commonness of certain rare cell types in the peripheral blood under physiological conditions. Thus, the isolation and detection of circulating rare cells appears to be merely a technological problem. Knowledge about rare cell types that may circulate the blood stream will help to advance the field of cell-based liquid biopsy by supporting inter-platform comparability, making use of biological correct cutoffs and "mining" new biomarkers and combinations thereof in clinical diagnosis and therapy. Therefore, this review intends to lay ground for a comprehensive analysis of the peripheral blood rare cell population given the necessity to target a broader range of cell types for improved biomarker performance in cell-based liquid biopsy.

An update of circulating rare cell types in healthy adult peripheral blood: findings of immature erythroid precursors.

BACKGROUND: Circulating rare cells (CRCs) are benign or malignant minuscule events in the peripheral blood or other bodily fluids. The detection and quantification of certain CRC types is an invaluable or proposed candidate biomarker for diagnosis, prognosis and prediction of various pathological conditions. The list of CRC types and biomarker applicability thereof continues to expand along with improvements in cell selection technology. Past findings may suggest commonness of healthy donor peripheral blood circulating mature erythroblasts. This work suggests the occurrence of morphologically distinct bone marrow native circulating early erythroid precursors that we intend to add to the list of CRCs. METHODS: We tested 15 healthy individuals that varied in age and gender employing a negative cell selection assay based on magnetic bead technology to characterize healthy adult circulating CD45 negative cell events using cell surface markers CD71 and glycophorin-A. RESULTS: Positive events were detected and varied in cell and nuclear size ranging between 7.5 µm till 15 µm and 4.5 till 9.2 µm, respectively with distinct appearance under bright field microscope. Cell rarity increased with cell and nuclear size. Largest cells exceeded 13.5 µm in cell diameter and were found in 7 out of 15 donors. CONCLUSIONS: Circulating erythroid precursors occur at different stages of maturation and may be part of the benign CRC spectrum.

Aneuploid CTC and CEC.

Conventional circulating tumor cell (CTC) detection technologies are restricted to large tumor cells (> white blood cells (WBCs)), or those unique carcinoma cells with double positive expression of surface epithelial cell adhesion molecule (EpCAM) for isolation, and intracellular structural protein cytokeratins (CKs) for identification. With respect to detecting the full spectrum of highly heterogeneous circulating rare cells (CRCs), including CTCs and circulating endothelial cells (CECs), it is imperative to develop a strategy systematically coordinating all tri-elements of nucleic acids, biomarker proteins, and cellular morphology, to effectively enrich and comprehensively identify CRCs. Accordingly, a novel strategy integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), independent of cell size variation and free of hypotonic damage as well as anti-EpCAM perturbing, has been demonstrated to enable in situ phenotyping multi-protein expression, karyotyping chromosome aneuploidy, and detecting cytogenetic rearrangements of the ALK gene in non-hematologic CRCs. Symbolic non-synonymous single nucleotide variants (SNVs) of both the TP53 gene (P33R) in each single aneuploid CTCs, and the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor suppressor gene in each examined aneuploid CECs, were identified for the first time across patients with diverse carcinomas. Comprehensive co-detecting observable aneuploid CTCs and CECs by SE-iFISH, along with applicable genomic and/or proteomic single cell molecular profiling, are anticipated to facilitate elucidating how those disparate categories of aneuploid CTCs and CECs cross-talk and functionally interplay with tumor angiogenesis, therapeutic drug resistance, tumor progression, and cancer metastasis.