Detection of PD­L1 expression and epithelial­mesenchymal transition of circulating tumor cells in non­small cell lung cancer.

The present study aimed to assess the roles of peripheral circulating tumor cell (CTC) count, CTC subtypes and programmed death ligand 1 (PD-L1) expression in the clinical staging and prognosis of patients with non-small cell lung cancer (NSCLC). A total of 100 patients with NSCLC with available tumor tissues were enrolled in the present study, and 7.5 ml peripheral blood was collected. Patients were divided into PD-L1-positive and PD-L1-negative groups according to PD-L1 immunohistochemical staining. Peripheral blood samples from both groups were analyzed to determine the CTC count, epithelial-type CTCs (E-CTCs), mesenchymal-type CTCs (M-CTCs) and PD-L1 expression. Clinical data were collected, and patients were followed up for a maximum of 36 months, with patient death as the endpoint event. Patients with PD-L1-positive tumors had a worse prognosis compared with those with PD-L1-negative tumors (P=0.045). The PD-L1-positive group exhibited significantly higher numbers of CTCs and M-CTCs compared with the PD-L1-negative group (P≤0.05). However, the number of E-CTCs did not differ significantly between the two groups (P>0.05). PD-L1-positive patients with higher CTC and M-CTC counts had relatively poorer prognoses (P≤0.05), while the number of E-CTCs had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the CTC count (P≤0.05), while E-CTC and M-CTC counts did not significantly differ between the two groups (P>0.05). The PD-L1-positive group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs compared with the PD-L1-negative group (P≤0.05), while PD-L1+ E-CTC counts did not differ significantly between the two groups (P>0.05). The PD-L1-positive patients with a higher number of PD-L1+ CTCs and PD-L1+ M-CTCs had relatively poorer prognoses (P≤0.05), while the PD-L1+ E-CTC count had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs (P≤0.05), while PD-L1+ E-CTC counts did not significantly differ between the two groups (P>0.05). Based on univariate and multivariate analyses, the number of PD-L1+ M-CTCs was identified as an independent prognostic factor for NSCLC. In conclusion, the presence of CTCs in peripheral blood, particularly PD-L1+ M-CTC subtype, indicated poorer clinical staging and prognosis in patients with NSCLC. These findings suggested that CTCs, specifically the PD-L1+ M-CTC subtype, could serve as a monitoring indicator for the clinical staging and prognosis of patients with NSCLC.

Liquid biopsy: An arsenal for tumour screening and early diagnosis.

Cancer has become the second leading cause of death in the world, and more than 50% of cancer patients are diagnosed at an advanced stage. Early diagnosis of tumours is the key to improving patient quality of life and survival time and reducing the socioeconomic burden. However, there is still a lack of reliable early diagnosis methods in clinical practice. In recent years, liquid biopsy technology has developed rapidly. It has the advantages of noninvasiveness, easy access to sample sources, and reproducibility. It has become the main focus of research on the early diagnosis methods of tumours. This review summarises the research progress of existing liquid biopsy markers, such as circulating tumour DNA, circulating viral DNA, DNA methylation, circulating tumour cells, circulating RNA, exosomes, and tumour education platelets in early diagnosis of tumours, and analyses the current advantages and limitations of various markers, providing a direction for the application and transformation of liquid biopsy research in early diagnosis of clinical tumours.

Characterisation of circulating tumor-associated and immune cells in patients with advanced-stage non-small cell lung cancer.

Objectives: Globally, non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and the leading cause of cancer-related deaths. Tumor-associated circulating cells in NSCLC can have a wide variety of morphological and phenotypic characteristics, including epithelial, immunological or hybrid subtypes. The distinctive characteristics and potential clinical significance of these cells in patients with NSCLC are explored in this study. Methods: We utilised a spiral microfluidic device to enrich large cells and cell aggregates from the peripheral blood samples of NSCLC patients. These cells were characterised through high-resolution immunofluorescent imaging and statistical analysis, correlating findings with clinical information from our patient cohort. Results: We have identified varied populations of heterotypic circulating tumor cell clusters with differing immune cell composition that included a distinct class of atypical tumor-associated macrophages that exhibits unique morphology and cell size. This subtype's prevalence is positively correlated with the tumor stage, progression and metastasis. Conclusions: Our study reveals a heterogeneous landscape of circulating tumor cells and their clusters, underscoring the complexity of NSCLC pathobiology. The identification of a unique subtype of atypical tumor-associatedmacrophages that simultaneously express both tumor and immune markers and whose presence correlates with late disease stages, poor clinical outcomes and metastatic risk infers  the potential of these cells as biomarkers for NSCLC staging and prognosis. Future studies should focus on the role of these cells in the tumor microenvironment and their potential as therapeutic targets. Additionally, longitudinal studies tracking these cell types through disease progression could provide further insights into their roles in NSCLC evolution and response to treatment.

Anchorage Dependence and Cancer Metastasis.

The process of cancer metastasis is dependent on the cancer cells' capacity to detach from the primary tumor, endure in a suspended state, and establish colonies in other locations. Anchorage dependence, which refers to the cells' reliance on attachment to the extracellular matrix (ECM), is a critical determinant of cellular shape, dynamics, behavior, and, ultimately, cell fate in nonmalignant and cancer cells. Anchorage-independent growth is a characteristic feature of cells resistant to anoikis, a programmed cell death process triggered by detachment from the ECM. This ability to grow and survive without attachment to a substrate is a crucial stage in the progression of metastasis. The recently discovered phenomenon named "adherent-to-suspension transition (AST)" alters the requirement for anchoring and enhances survival in a suspended state. AST is controlled by four transcription factors (IKAROS family zinc finger 1, nuclear factor erythroid 2, BTG anti-proliferation factor 2, and interferon regulatory factor 8) and can detach cells without undergoing the typical epithelial-mesenchymal transition. Notably, AST factors are highly expressed in circulating tumor cells compared to their attached counterparts, indicating their crucial role in the spread of cancer. Crucially, the suppression of AST substantially reduces metastasis while sparing primary tumors. These findings open up possibilities for developing targeted therapies that inhibit metastasis and emphasize the importance of AST, leading to a fundamental change in our comprehension of how cancer spreads.

Two-color diffuse in vivo flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.

Liquid biopsy for human cancer: cancer screening, monitoring, and treatment.

Currently, tumor treatment modalities such as immunotherapy and targeted therapy have more stringent requirements for obtaining tumor growth information and require more accurate and easy-to-operate tumor information detection methods. Compared with traditional tissue biopsy, liquid biopsy is a novel, minimally invasive, real-time detection tool for detecting information directly or indirectly released by tumors in human body fluids, which is more suitable for the requirements of new tumor treatment modalities. Liquid biopsy has not been widely used in clinical practice, and there are fewer reviews of related clinical applications. This review summarizes the clinical applications of liquid biopsy components (e.g., circulating tumor cells, circulating tumor DNA, extracellular vesicles, etc.) in tumorigenesis and progression. This includes the development process and detection techniques of liquid biopsies, early screening of tumors, tumor growth detection, and guiding therapeutic strategies (liquid biopsy-based personalized medicine and prediction of treatment response). Finally, the current challenges and future directions for clinical applications of liquid biopsy are proposed. In sum, this review will inspire more researchers to use liquid biopsy technology to promote the realization of individualized therapy, improve the efficacy of tumor therapy, and provide better therapeutic options for tumor patients.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis.

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

Programmed Death Ligand 1 Expression in Circulating Tumor Cells as a Predictor and Monitor of Response to Atezolizumab plus Bevacizumab Treatment in Patients with Hepatocellular Carcinoma.

There remains no reliable biomarker of therapeutic efficacy in hepatocellular carcinoma (HCC) for the PD-L1 inhibitor atezolizumab and bevacizumab (Atezo/Bev). Circulating tumor cells (CTCs) enable the serial collection of living tumor cells. Pre-treatment and serial CTC gene expression changes and tumor histology were evaluated to identify predictors of response to Atezo/Bev. Peripheral blood from 22 patients with HCC treated with Atezo/Bev and 24 patients treated with lenvatinib was serially collected. The RNA expression in CTCs was analyzed using qRT-PCR. Higher PD-L1 expression in pre-treatment CTCs was associated with response and improved prognosis with Atezo/Bev treatment, but not with lenvatinib. There was no correlation between PD-L1 expression in CTCs and that in liver tumor biopsy specimens scored using imaging software. Furthermore, PD-L1 RNA expression in CTCs was dynamically altered by Atezo/Bev, decreasing during effective response and increasing upon progression. CTC-derived RNA collected during Atezo/Bev indicates that patients with higher PD-L1 expression in CTCs at baseline were 3.9 times more responsive to treatment. Therefore, PD-L1 RNA levels in CTCs are an accurate response predictor and may be a monitorable biomarker that changes dynamically to reflect the response during Atezo/Bev treatment.

High-throughput enrichment of portal venous circulating tumor cells for highly sensitive diagnosis of CA19-9-negative pancreatic cancer patients using inertial microfluidics.

The carbohydrate antigen 19-9 (CA19-9) is commonly used as a representative biomarker for pancreatic cancer (PC); however, it lacks sensitivity and specificity for early-stage PC diagnosis. Furthermore, some patients with PC are negative for CA19-9 (<37 U/mL), which introduces additional limitations to their accurate diagnosis and treatment. Hence, improved methods to accurately detect PC stages in CA19-9-negative patients are warranted. In this study, tumor-proximal liquid biopsy and inertial microfluidics were coupled to enable high-throughput enrichment of portal venous circulating tumor cells (CTCs) and support the effective diagnosis of patients with early-stage PC. The proposed inertial microfluidic system was shown to provide size-based enrichment of CTCs using inertial focusing and Dean flow effects in slanted spiral channels. Notably, portal venous blood samples were found to have twice the yield of CTCs (21.4 cells per 5 mL) compared with peripheral blood (10.9 CTCs per 5 mL). A combination of peripheral and portal CTC data along with CA19-9 results showed to greatly improve the average accuracy of CA19-9-negative PC patients from 47.1% with regular CA19-9 tests up to 87.1%. Hence, portal venous CTC-based microfluidic biopsy can be used with high sensitivity and specificity for the diagnosis of early-stage PC, particularly in CA19-9-negative patients.

Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines.

BACKGROUND: Circulating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters. METHODS: The assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used. RESULTS: We systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC. CONCLUSIONS: The study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.

The correlation study between TOP2A gene expression in circulating tumor cells and chemotherapeutic drug resistance of patients with breast cancer.

BACKGROUND: Patients with breast cancer (BC) at advanced stages have poor outcomes because of high rate of recurrence and metastasis. Biomarkers for predicting prognosis remain to be explored. This study aimed to evaluate the relationships between circulating tumor cells (CTCs) and outcomes of BC patients. PATIENTS AND METHODS: A total of 50 female were enrolled in this study. Their diagnoses were determined by clinical characteristics, image data, and clinical pathology. CTC subtypes and TOP2A gene expression on CTCs were detected by CanPatrol™ technology and triple color in situ RNA hybridization (RNA-ISH), which divided into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and hybrid CTCs (HCTCs) based on their surface markers. Hormone receptor, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, was measured by immunohistochemistry (IHC) method before treatment. The risk factors for predicting recurrence and metastasis were calculated by COX risk regression model. The progression-free survival (PFS) of patients was determined using Kaplan-Meier survival curve. RESULTS: The patients with a large tumor size (≥ 3 cm) and advanced tumor node metastasis (TNM) stages had high total CTCs (TCTCs) (P < 0.05). These patients also had high TOP2A expression level. COX risk regression analysis indicated that TOP2A expression levels in TCTCs, ER + , HER-2 + , and TNM stages were critical risk factors for recurrence and metastasis of patients (P < 0.05). The PFS of patients with ≥ 5 TCTCs, ≥ 3 HCTCs, and positive TOP2A expression in ≥ 3 TCTCs was significantly longer than that in patient with < 5 TCTCs, < 3 HCTCs, and TOP2A expression in < 3 TCTCs (P < 0.05). In contrast, the PFS of patients with positive hormone receptors (ER + , PR + , HER-2 +) also was dramatically lived longer than that in patients with negative hormone receptor expression. CONCLUSIONS: High TCTC, HCTCs, and positive TOP2A gene expression on CTCs were critical biomarkers for predicting outcomes of BC patients. Positive hormone receptor expression in BC patients has significant favor PFS.

Octamer-binding transcription factor 4-positive circulating tumor cell predicts worse treatment response and survival in advanced cholangiocarcinoma patients who receive immune checkpoint inhibitors treatment.

BACKGROUND: Octamer-binding transcription factor 4-positive circulating tumor cell (OCT4+CTC) exhibits high stemness and invasive potential, which may influence the efficacy of immune checkpoint inhibitors (ICI). This study aimed to assess the prognostic role of OCT4+CTC in advanced cholangiocarcinoma (CCA) patients who received ICI treatment. METHODS: In total, 40 advanced CCA patients who received ICI treatment were included, and CTC and OCT4 counts were detected via a Canpatrol system and an RNA in situ hybridization method before ICI treatment. Patients were subsequently divided into none CTC, OCT4-CTC, and OCT4+CTC groups. Patients were followed up for a median of 10.4 months. RESULTS: The percentages of patients in none CTC, OCT4-CTC, and OCT4+CTC groups were 25.0%, 30.0%, and 45.0%, respectively. The proportion of patients with lymph node metastasis was highest in OCT4+CTC group, followed by none CTC group, and lowest in OCT4-CTC group (P = 0.025). The objective response rate (ORR) was lowest in OCT4+CTC group, moderate in OCT4-CTC group, and highest in none CTC group (P = 0.009), while disease control rate was not different among three groups (P = 0.293). In addition, progression-free survival (PFS) (P < 0.001) and overall survival (OS) (P = 0.001) were shorter in the OCT4+CTC group than in none CTC & OCT4-CTC group. Moreover, OCT4+CTC (versus none CTC) was independently linked with poorer PFS [hazard ratio (HR) = 6.752, P = 0.001] and OS (HR = 6.674, P = 0.003) in advanced CCA patients. CONCLUSION: OCT4+CTC relates to lymph node metastasis and shows a good predictive value for poor treatment response and survival in advanced CCA patients who receive ICI treatment.

Enumeration and Molecular Characterization of Circulating Tumor Cell Using an Epithelial Cell Adhesion Molecule/Vimentin/Epidermal Growth Factor Receptor Joint Capture System in Lung Cancer.

Background: Detection rate and isolation yield of circulating tumor cells (CTCs) are low in lung cancer with approaches due to CTC invasiveness and heterogeneity. In this study, on the basis of the epithelial cell adhesion molecule (EpCAM) phenotype, markers of vimentin and epidermal growth factor receptor (EGFR) phenotype were added to jointly construct a precise and efficient CTC capture system for capture of lung cancer CTCs. Methods: A CTC capture system combined with EpCAM lipid magnetic bead (Ep-LMB)/vimentin lipid magnetic bead (Vi-LMB)/EGFR lipid magnetic bead (EG-LMB) was constructed, and its performance was tested. The amount of CTC captured in the blood of patients with lung cancer was detected by immunofluorescence identification and analyzed for clinical relevance. Results: The constructed CTC capture system has low cytotoxicity. The capture efficiency of lung cancer cells in phosphate belanced solution (PBS) system was 95.48%. The capture efficiency in the blood simulation system is 94.55%. The average number of CTCs in the blood of patients with lung cancer was 9.73/2 mL. The quantity distribution of CTCs is significantly correlated with tumor staging and metastasis. The area under the curve (AUC) of CTCs for the diagnosis of lung cancer was 0.9994 (95% CI = 0.9981-1.000, P < .0001). The cutoff value was 4.5/2 mL. The sensitivity was 99.39%, and the specificity was 96.88%. Conclusion: The EpCAM/vimentin/EGFR combined capture system has feasibility and high sensitivity in the detection of lung cancer CTC typing, which can be used as an auxiliary diagnostic indicator for lung cancer and is expected to promote the clinical application of CTCs.

Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

Significance: We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. Aim: In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). Approach: A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. Results: The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. Conclusions: The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Is circulating tumor cell count-driven cost-effective for first-line therapy choice in HR+/HER2- metastatic breast cancer in the United States?

BACKGROUND: Circulating tumor cell (CTC) counting may be a useful non-invasive biomarker that helps patients choose first-line treatment options. Nevertheless, the cost of CTC inspection may impose an economic burden on patients, necessitating the simultaneous consideration of both its clinical effectiveness and cost. We evaluated the cost-effectiveness of CTC count-guided chemotherapy and endocrine therapy as first-line therapy for HR+/HER2-metastatic breast cancer (MBC) from the perspective of US payers. METHODS: Based on the STIC CTC trial, a Markov model was constructed for three health states, and health outcomes were measured in quality-adjusted life years (QALYs) and incremental cost-effectiveness ratio (ICER). One-way and probabilistic sensitivity analyses were performed to assess the robustness of the incremental cost per QALY. RESULTS: The base-case analysis revealed that CTC count-driven treatment was associated with improved effectiveness by 0.07 QALYs and increased the overall cost by $9187.05 compared with clinician-driven first-line treatment choices, leading to an ICER of $138 354.15 per QALY. One-way sensitivity analysis indicated that the model was most sensitive to the cost of treatment for neutropenia and the utility for PFS; probability sensitivity analysis indicated that CTC count-driven treatment choices would be considered the cost-effective option at a willingness-to-pay threshold of $150 000 per QALY. CONCLUSIONS: The findings of this cost-effectiveness analysis suggest that, at the current price of CTC enumeration, choosing first-line treatment options based on CTC count is a cost-effectiveness approach for treating patients with HR+/HER2- MBC in the US.

Functional requirement of alternative splicing in epithelial-mesenchymal transition of pancreatic circulating tumor.

Circulating tumor cells (CTCs) that undergo epithelial-to-mesenchymal transition (EMT) can provide valuable information regarding metastasis and potential therapies. However, current studies on the EMT overlook alternative splicing. Here, we used single-cell full-length transcriptome data and mRNA sequencing of CTCs to identify stage-specific alternative splicing of partial EMT and mesenchymal states during pancreatic cancer metastasis. We classified definitive tumor and normal epithelial cells via genetic aberrations and demonstrated dynamic changes in the epithelial-mesenchymal continuum in both epithelial cancer cells and CTCs. We provide the landscape of alternative splicing in CTCs at different stages of EMT, uncovering cell-type-specific splicing patterns and splicing events in cell surface proteins suitable for therapies. We show that MBNL1 governs cell fate through alternative splicing independently of changes in gene expression and affects the splicing pattern during EMT. We found a high frequency of events that contained multiple premature termination codons and were enriched with C and G nucleotides in close proximity, which influence the likelihood of stop codon readthrough and expand the range of potential therapeutic targets. Our study provides insights into the EMT transcriptome's dynamic changes and identifies potential diagnostic and therapeutic targets in pancreatic cancer.

Training an automated circulating tumor cell classifier when the true classification is uncertain.

Circulating tumor cell (CTC) and tumor-derived extracellular vesicle (tdEV) loads are prognostic factors of survival in patients with carcinoma. The current method of CTC enumeration relies on operator review and, unfortunately, has moderate interoperator agreement (Fleiss' kappa 0.60) due to difficulties in classifying CTC-like events. We compared operator review, ACCEPT automated image processing, and refined the output of a deep-learning algorithm to identify CTC and tdEV for the prediction of survival in patients with metastatic and nonmetastatic cancers. Operator review is only defined for CTC. Refinement was performed using automatic contrast maximization CM-CTC of events detected in cancer and in benign samples (CM-CTC). We used 418 samples from benign diseases, 6,293 from nonmetastatic breast, 2,408 from metastatic breast, and 698 from metastatic prostate cancer to train, test, optimize, and evaluate CTC and tdEV enumeration. For CTC identification, the CM-CTC performed best on metastatic/nonmetastatic breast cancer, respectively, with a hazard ratio (HR) for overall survival of 2.6/2.1 vs. 2.4/1.4 for operator CTC and 1.2/0.8 for ACCEPT-CTC. For tdEV identification, CM-tdEV performed best with an HR of 1.6/2.9 vs. 1.5/1.0 with ACCEPT-tdEV. In conclusion, contrast maximization is effective even though it does not utilize domain knowledge.

Phenotypic Transitions the Processes Involved in Regulation of Growth and Proangiogenic Properties of Stem Cells, Cancer Stem Cells and Circulating Tumor Cells.

Epithelial-mesenchymal transition (EMT) is a crucial process with significance in the metastasis of malignant tumors. It is through the acquisition of plasticity that cancer cells become more mobile and gain the ability to metastasize to other tissues. The mesenchymal-epithelial transition (MET) is the return to an epithelial state, which allows for the formation of secondary tumors. Both processes, EMT and MET, are regulated by different pathways and different mediators, which affects the sophistication of the overall tumorigenesis process. Not insignificant are also cancer stem cells and their participation in the angiogenesis, which occur very intensively within tumors. Difficulties in effectively treating cancer are primarily dependent on the potential of cancer cells to rapidly expand and occupy secondarily vital organs. Due to the ability of these cells to spread, the concept of the circulating tumor cell (CTC) has emerged. Interestingly, CTCs exhibit molecular diversity and stem-like and mesenchymal features, even when derived from primary tumor tissue from a single patient. While EMT is necessary for metastasis, MET is required for CTCs to establish a secondary site. A thorough understanding of the processes that govern the balance between EMT and MET in malignancy is crucial.

Detection of Circulating Tumor Cells by Cell Block Technique in Malignant Tumors.

Introduction: Cancer is a leading cause of death worldwide and is a major cause of morbidity. To deal with this magnitude of cancers and their diagnostic and prognostics, a multitude of prognostic biomarkers for various cancers have been explored over the decades, with detection of circulating tumor cells (CTCs) in the peripheral blood being one of them. This study was undertaken to explore the routine procedure of cell block in the cytopathology lab to isolate and detect CTCs. Materials and Methods: In this cross-sectional study, 112 peripheral blood samples sent for routine blood investigations of various cancer patients were utilized for the preparation of cell block. The sections from the cell block were stained routinely and evaluated for the presence of CTCs. The statistical analysis was done using Mac Statplus software version 8.0. Results: The malignancies were tabulated as per the International Classification of Diseases for Oncology, third edition (ICD-O-3). The maximum number of cases were from C 50 (breast) - 41/112 (36.6%), followed by C15-C26 (Digestive organs) - 19/112 (16.9%), and C00-C14 (lip, oral cavity, and pharynx) - 18/112 (16.07%) cases. CTC was detected in six (5.35%) out of 112 cases, out of which three were from the breast and one each from category C6.9 (mouth), C32.0 (glottis), and C53.8 (cervix uteri). Conclusion: Among various advanced and molecular techniques available for the detection of CTCs, the cell block technique proves to be one of the effective methods, especially in resource-limited settings as these can further be utilized for additional diagnostic techniques similar to the ones employed for routine paraffin blocks.

Tumor-educated platelets.

Beyond traditional roles in homeostasis and coagulation, growing evidence suggests that platelets also reflect malignant transformation in cancer. Platelets are present in the tumor microenvironment where they interact with cancer cells. This interaction results in direct and indirect "education" as evident by platelet alterations in adhesion molecules, glycoproteins, nucleic acids, proteins and various receptors. Subsequently, these tumor-educated platelets (TEPs) circulate throughout the body and play pivotal roles in promotion of tumor growth and dissemination. Accordingly, platelet status can be considered a unique blood-based biomarker that can potentially predict prognosis and therapeutic success. Recently, liquid biopsies including TEPs have received much attention as safe, minimally invasive and sensitive alternatives for patient management. Herein, we provide an overview of TEPs and explore their benefits and limitations in cancer.