Biphasic co-detection of melanoma aneuploid tumor cells and tumor endothelial cells in guidance of specifying the field cancerized surgical excision margin and administering immunotherapy.

An optimum safety excision margin (EM) delineated by precise demarcation of field cancerization along with reliable biomarkers that enable predicting and timely evaluating patients' response to immunotherapy significantly impact effective management of melanoma. In this study, optimized biphasic "immunofluorescence staining integrated with fluorescence insitu hybridization" (iFISH) was conducted along the diagnosis-metastasis-treatment-cellular MRD axis to longitudinally co-detect a full spectrum of intact CD31- aneuploid tumor cells (TCs), CD31+ aneuploid tumor endothelial cells (TECs), viable and necrotic circulating TCs (CTCs) and circulating TECs (CTECs) expressing PD-L1, Ki67, p16 and Vimentin in unsliced specimens of the resected primary tumor, EM, dissected sentinel lymph nodes (SLNs) and peripheral blood in an early-stage melanoma patient. Numerous PD-L1+ aneuploid TCs and TECs were detected at the conventional safety EM (2 cm), quantitatively indicating the existence of a field cancerized EM for the first time. Contrary to highly heterogeneous PD-L1 expression and degrees of Chr8 aneuploidy in TCs and TECs in the primary lesions as well as CTCs and CTECs in peripheral blood, almost all TCs and TECs in SLNs and EM were homogeneously PD-L1+ haploid cells. Dynamic monitoring and cellular MRD assessment revealed that, in contrast to PD-L1+ CTCs being responsive to the immune checkpoint inhibitor (ICI-anti-PD-1), multiploid (≥pentasomy 8) PD-L1+ and Ki67+ CTECs were respectively resistant to ICI-sensitized T cells. In therapeutically stressed lymphatic and hematogenous metastatic cascades, stratified phenotypic and karyotypic profiling of iFISH tissue and liquid biopsied TCs, TECs, CTCs and CTECs in future large-cohort studies will enable appropriate re-specification of the optimal safety EM and distribution mapping of in-depth characterized, subcategorized target cells to help illustrate their metastatic relevance, ultimately improving risk stratification and clinical intervention of tumor progression, metastases, therapy resistance and cancer relapse.

The Presence of Small-Size Circulating Tumor Cells Predicts Worse Prognosis in Non-Small Cell Lung Cancer Patients.

CONTEXT.­: Most patients with non-small cell lung cancers (NSCLC) are diagnosed at advanced stages. The 5-year survival rate of patients with advanced lung cancer is less than 20%, which makes lung cancer the leading cause of cancer-related deaths worldwide. OBJECTIVE.­: To identify indicators that can predict the prognosis of lung cancer patients. DESIGN.­: To determine the correlation between circulating tumor cells (CTCs), circulating tumor-derived endothelial cells (CTECs), and their subtypes and the prognosis of patients with NSCLC, 80 patients with lung cancer were recruited and 48 patients who met the enrollment criteria were selected in this study. Peripheral blood was collected from the enrolled patients before any treatment and analyzed by the subtraction enrichment and immunostaining-fluorescence in situ hybridization technique to determine the correlation between CTCs and CTECs and lung cancer disease progression and to identify prognostic indicators. RESULTS.­: In all patients, the positive rate of CTCs was 100% and the positive rate of CTECs was 81.3%. The CTEC positivity rate was higher in late-stage patients than in early-stage patients (P = .03). Patients with advanced or lymph node metastases had a higher rate of small-size CTC positivity than those with early or no lymph node metastases. Large-size CTEC positivity was higher in patients with advanced NSCLC than in early-stage patients. Patients with ≥1 small-size CTC had shorter progression-free survival, and it was an independent prognostic factor. CONCLUSIONS.­: Small-size CTCs are a reliable prognostic indicator and a probable predictor of the severity of disease in NSCLC patients.

Longitudinal detection of subcategorized CD44v6+ CTCs and circulating tumor endothelial cells (CTECs) enables novel clinical stratification and improves prognostic prediction of small cell lung cancer: A prospective, multi-center study.

Current management of small cell lung cancer (SCLC) remains challenging. Effective biomarkers are needed to subdivide patients presenting distinct treatment response and clinical outcomes. An understanding of heterogeneous phenotypes of aneuploid CD31- circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells (CTECs) may provide novel insights in the clinical management of SCLC. In the present translational and prospective study, increased cancer metastasis-related cell proliferation and motility, accompanied with up-regulated mesenchymal marker vimentin but down-regulated epithelial marker E-cadherin, were observed in both lentivirus infected SCLC and NSCLC cells overexpressing the stemness marker CD44v6. Aneuploid CTCs and CTECs expressing CD44v6 were longitudinally detected by SE-iFISH in 120 SCLC patients. Positive detection of baseline CD44v6+ CTCs and CD44v6+ CTECs was significantly associated with enhanced hepatic metastasis. Karyotype analysis revealed that chromosome 8 (Chr8) in CD44v6+ CTCs shifted from trisomy 8 towards multiploidy in post-therapeutic patients compared to pre-treatment subjects. Furthermore, the burden of baseline CD44v6+ CTCs (t0) or amid the therapy (t1-2), the ratio of baseline CD31+ CTEC/CD31- CTC (t0), and CTC-WBC clusters (t0) were correlated with treatment response and distant metastases, particularly brain metastasis, in subjects with limited disease (LD-SCLC) but not in those with extensive disease (ED-SCLC). Multivariate survival analysis validated that longitudinally detected CD44v6+/CD31- CTCs was an independent prognostic factor for inferior survival in SCLC patients. Our study provides evidence for the first time that comprehensive analyses of CTCs, CTECs, and their respective CD44v6+ subtypes enable clinical stratification and improve prognostic prediction of SCLC, particularly for potentially curable LD-SCLC.

Post-therapeutic circulating tumor cell-associated white blood cell clusters predict poor survival in patients with advanced driver gene-negative non-small cell lung cancer.

PURPOSE: This study aimed to investigate the clinical utility of diverse aneuploid circulating tumor cell (CTC) subtypes and particularly CTC-associated white blood cell (CTC-WBC) clusters in predicting treatment response, prognosis and real-time monitoring disease progression in advanced driver gene-negative non-small lung cancer (NSCLC) patients. MATERIALS AND METHODS: A total of 74 eligible patients were prospectively enrolled and serial blood samples were collected at pre-treatment(t0), after two cycles of therapy (t1) and at post-four-to-six treatment cycles (t2). Co-detection of diverse subtypes of aneuploid CTCs and CTC-WBC clusters was conducted in advanced NSCLC patients receiving first-line treatment. RESULTS: At baseline, CTCs were detected in 69 (93.24%) patients and CTC-WBC clusters were detected in 23 (31.08%) patients. Patients with CTCs < 5/6ml or with CTC-WBC clusters undetectable exhibited a better treatment response than patients with pre-therapeutic aneuploid CTCs ≥ 5/6ml or harboring CTC-WBC clusters (p = 0.034 and p = 0.012, respectively). Before treatment, patients bearing tetraploid CTCs ≥ 1/6ml showed significantly inferior progression-free survival (PFS) [hazard ratio (HR):2.420, 95% confidence interval (CI): 1.426-4.106; p = 0.001] and overall survival (OS) compared to patients with tetraploid CTCs < 1/6ml (HR:1.907, 95%CI: 1.119-3.251; p = 0.018). A longitudinal study demonstrated that post-therapeutic patients harboring CTC-WBC clusters displayed the reduced PFS and OS compared with those without CTC-WBC clusters, and subgroup analysis showed that the presence of CTC-WBC clusters indicated a worse prognosis in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. After adjusting for multiple significant factors, post-therapeutic CTC-WBC clusters were the only independent predictor of both PFS (HR:2.872, 95% CI: 1.539-5.368; p = 0.001) and OS (HR:2.162, 95% CI: 1.168-4.003; p = 0.014). CONCLUSIONS: In addition to CTCs, longitudinal detection of CTC-WBC clusters provided a feasible tool to indicate initial treatment response, dynamically monitor disease progression and predict survival in driver gene-negative advanced NSCLC patients.

Case report: Post-therapeutic laryngeal carcinoma patient possessing a high ratio of aneuploid CTECs to CTCs rapidly developed de novo malignancy in pancreas.

Effectively evaluating therapeutic efficacy, detecting minimal residual disease (MRD) after therapy completion, and predicting early occurrence of malignancy in cancer patients remain as unmet imperative clinical demands. This article presents a case of a laryngeal carcinoma patient who had a surgical resection and complete post-operative chemoradiotherapy in combination with the targeted therapy, then rapidly developed pancreatic adenocarcinoma. Detected by SE-iFISH, the patient had a substantial amount of 107 non-hematological aneuploid circulating rare cells including 14 circulating tumor cells (CTCs, CD31-/CD45-) and 93 circulating tumor endothelial cells (CTECs, CD31+/CD45-) with a high ratio of CTECs/CTCs > 5 upon finishing post-surgical combination regimens. Positive detection of those aneuploid non-hematological circulating rare cells was five months prior to subsequent plasma CA19-9 increasing and ten months before the de novo pancreatic cancer was diagnosed by medical imaging modalities. Besides previously reported clinical utilities of co-detection of aneuploid CD31- CTCs and CD31+ CTECs in real-time evaluation of therapeutic efficacy, longitudinal monitoring of emerging treatment resistance and adequate detection of MRD, a large cohort study is necessary to further investigate whether, and how, a high ratio of MRD CTECs to CTCs may function as an appropriate index forecasting either occurrence or metastatic distant recurrence of malignancy in post-therapeutic cancer patients.

Nonhematogenic circulating aneuploid cells confer inferior prognosis and therapeutic resistance in gliomas.

Aneuploidy is the hallmark of malignancy. Our previous study successfully detected nonhematogenic circulating aneuploidy cells (CACs) in types of gliomas. The current prospective clinical study aims to further precisely subcategorize aneuploid CACs, including CD31- circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells, and thoroughly investigate the clinical utilities of these different subtypes of cells. Co-detection and analysis of CTCs and circulating tumor-derived endothelial cells (CTECs) expressing CD133, glial fibrillary acidic protein (GFAP), or epidermal growth factor receptor variant III (EGFR vIII) were performed by integrated subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) in 111 preoperative primary diffuse glioma patients. Aneuploid CACs could be detected in most de novo glioma patients. Among detected CACs, 45.6% were CD31- /CD45- aneuploid CTCs and the remaining 54.4% were CD31+ /CD45- aneuploid CTECs. Positive detection of CTECs significantly correlated with disruption of the blood-brain barrier. The median number of large CTCs (L CTCs, >5 µm, 2) in low-grade glioma (WHO grade 2) was less than high-grade glioma (WHO grades 3 and 4) (3, p = 0.044), but this difference was not observed in small CTCs (S CTCs, ≤5 µm), CTECs or CACs (CTCs + CTECs). The numbers of CTCs, CTECs, or CACs in patients with contrast-enhancing (CE) lesions considerably exceeded that of non-CE lesions (p < 0.05). Receiver operating characteristic curves demonstrated that CD31+ CTECs, especially L CTECs, exhibited a close positive relationship with CE lesions. Survival analysis revealed that the high number of CD31- CTCs could be an adverse factor for compromised progression-free survival and overall survival. Longitudinal surveillance of CD31- CTCs was suitable for evaluating the therapeutic response and for monitoring potential emerging treatment resistance.

Analysis of the prognostic role and biological characteristics of circulating tumor cell-associated white blood cell clusters in non-small cell lung cancer.

Background: Recently, circulating tumor-cell-associated white blood cell (CTC-WBC) clusters have been reported to have prognostic value in some cancers. The prognostic role of CTC-WBC clusters in lung cancer has not yet been elucidated. Very little information is available about the biological characteristics of CTC-WBC clusters. Methods: A total of 82 patients with non-small cell lung cancer (NSCLC) were included in this study, and 61 patients with advanced-stage disease were closely followed-up. All patients had blood drawn prior to treatment. Subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) platform was used to isolate and identify CTCs and CTC-WBC clusters. Kaplan-Meier survival analysis and Cox regression analysis were applied to assess patient progression-free survival (PFS). Further, qualitative and quantitative analyses the size and ploidy characteristics of CTC-WBC clusters. Results: Firstly, CTC-WBC clusters appeared more in the advanced (stage III and IV) stage (P=0.043) than in the early stage. Furthermore, the multivariable analysis (Cox proportional hazards model) revealed that the high-CTC (≥7/6 mL) group and CTC-WBC clusters (≥1/6 mL) positive group both had significantly worse PFS, with a hazard ratio (HR) of 2.89 [95% confidence interval (CI): 1.36-6.17, P=0.006] and 2.18 (95% CI: 1.07-4.43, P=0.031), respectively. In the conjoint analysis, compared to patients with <7 CTCs/6 mL without CTC-WBC clusters, patients with ≥7 CTCs/6 mL with CTC-WBC clusters had the highest risk of progression (HR =7.13, 95% CI: 2.51-20.23, P<0.001). In addition, the presence of ≥3-cell CTC-WBC clusters in patients may indicate a shorter PFS (P<0.05) and a higher risk of progression (HR =2.90, 95% CI: 1.06-7.89, P=0.037). Furthermore, compared with the characteristics of the total CTCs, almost all of the CTCs that could recruit WBCs were large cells (≥5 µm) and exhibited polyploidy (≥ tetraploid) (both P<0.01). Conclusions: The presence of CTC-WBC clusters was an independent prognostic factor for advanced NSCLC. The joint analysis of CTCs and CTC-WBC clusters could provide additional prognostic value to the enumeration of CTCs alone. Besides, most of the CTCs in CTC-WBC clusters were large polyploid cells.

Identification and Comprehensive Co-Detection of Necrotic and Viable Aneuploid Cancer Cells in Peripheral Blood.

Aneuploid circulating tumor cells (CTCs, CD31-) and circulating tumor endothelial cells (CTECs, CD31+) exhibit an active interplay in peripheral blood, and play an essential role in tumorigenesis, neoangiogenesis, disease progression, therapy-resistant minimal residual disease (MRD), cancer metastasis and relapse. Currently, most CTC detection techniques are restricted to the indistinguishable quantification of circulating rare cells, including both necrotic and viable cells in cancer patients. Clinically imperative demands to distinguish and detect live and/or dead non-hematological aneuploid cancer cells in peripheral blood, which will assist in the rapid evaluation of therapeutic effects, real-time monitoring of treatment resistance longitudinally developed along with therapy and the effective detection of post-therapeutic MRD, have not yet been achieved. The integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH)-derived novel strategy was developed in this study, aiming to precisely identify and detect live and necrotic cancer cells (NC) enriched from carcinoma patients' biofluids. The innovative SE-iFISH (NC) provides a meaningful and practical approach to co-detect various viable and necrotic aneuploid CTCs and CTECs. The detected circulating rare cells can be characterized and categorized into diverse subtypes based upon cell viability, morphology, multiple tumor markers' expression, and the degree of aneuploidy relevant to both malignancy and therapeutic resistance. Each subtype of live or necrotic CTCs and CTECs possesses distinct utility in anti-cancer drug development, translational research, and clinical practice.

Aneuploid Circulating Tumor Cells as a Predictor of Response to Neoadjuvant Chemotherapy in Non-Small Cell Lung Cancer.

PURPOSE: This study aimed to explore the potential application of circulating tumor cells (CTCs) in predicting the therapeutic effect of neoadjuvant chemotherapy (NAC) in non-small-cell lung cancer (NSCLC). METHODS: Using integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization, the serial CTCs of patients with NSCLC were detected in 7.5 mL of blood at baseline and after two cycles of cisplatin-based NAC, and all aneuploidies of chromosome 8 were examined in the enriched CTCs. Tumor responses were evaluated radiologically with serial chest computed tomography (CT) using the response evaluation criteria in solid tumors and microscopically using the tumor cell necrosis rate (TCNR) of the resected specimen after NAC. RESULTS: After two cycles of cisplatin-based NAC, 89% (8/9) of the patients with radiological partial response to NAC had reduced CTC numbers, while 73% (8/11) of the patients with stable disease exhibited increased CTC numbers (P = 0.0098). On pathological examination, 90% (9/10) of patients with a TCNR lower than 30% had >1 CTC post-NAC, while 80% (4/5) of patients with a TCNR higher than 30% had ≤1 CTC post-NAC (P = 0.017). In aneuploidy analysis, the positive rate (CTC > 0) of triploid CTCs was found to have increased after NAC, in contrast with the tetraploid and multiploid CTCs. Furthermore, tetraploid and multiploid CTCs were found to be significantly downregulated in the patients with partial response to NAC. CONCLUSION: The correlations of aneuploid CTCs with both radiological and pathological responses in patients with NSCLC who received NAC were summarized, and the findings indicate that enumerating and karyotyping aneuploid CTCs can serve as a surrogate marker for disease monitoring in NSCLC.

Role of aneuploid circulating tumor cells and CD31+ circulating tumor endothelial cells in predicting and monitoring anti-angiogenic therapy efficacy in advanced NSCLC.

Prognosticating the efficacy of anti-angiogenic therapy through longitudinal monitoring and early detection of treatment resistance in cancer patients remain highly challenging. In this study, co-detection and comprehensive phenotypic and karyotypic molecular characterization of aneuploid circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) were conducted on non-small cell lung cancer (NSCLC) patients receiving bevacizumab plus chemotherapy. Prognostic values of the cell-based significant univariate risk factors identified by Cox regression analyses were progressively investigated. Subjects showing an increase in total post-therapeutic platelet endothelial cell adhesion molecule-1 (CD31)- CTCs and CD31+ CTECs exhibited a significantly reduced median progression-free survival (mPFS) and overall survival. Further stratification analyses indicated that pretherapeutic patients bearing vimentin (Vim)+ CTECs (mesenchymal M-type) at baseline revealed a significantly shortened mPFS compared with patients with Vim- CTECs. Post-therapeutic patients harboring epithelial cell adhesion molecule (EpCAM)+ CTCs and CTECs (epithelial E-type), regardless of Vim expression or not, showed a significantly reduced mPFS. Post-therapeutic patients possessing de novo EpCAM+ /Vim+ (hybrid E/M-type) CTECs displayed the shortest mPFS. Patients harboring either pre- or post-therapeutic EpCAM- /Vim- null CTECs (N-type) exhibited a better response to therapy compared to patients harboring EpCAM+ and/or Vim+ CTECs. The presented results support the notion that baseline Vim+ CTECs and post-therapeutic EpCAM+ CTCs and CTECs are predictive biomarkers for longitudinal monitoring of response to anti-angiogenesis combination regimens in NSCLC patients.

Dysregulated KRAS gene-signaling axis and abnormal chromatin remodeling drive therapeutic resistance in heterogeneous-sized circulating tumor cells in gastric cancer patients.

The mechanism by which heterogeneous-sized circulating tumor cells (CTCs) in gastric cancer (GC) patients are resistant to the targeted therapy and/or chemotherapy remains unclear. This study investigated prognostic value and genomic variations of size-heterogenous CTCs, in an attempt to unravel the molecular mechanisms underlying the therapeutic resistance, which is relevant to poor prognosis in GC. Aneuploid CTCs, detected in 111 advanced GC patients, were categorized into small (≤white blood cell [WBC], 25.54%) and large (>WBC, 74.46%) cells. Pre-treatment patients possessing ≥3 baseline small CTCs with trisomy 8 (SCTCstri) or ≥6 large multiploid CTCs (LCTCsmulti) showed an inferior median progression-free survival. Moreover, the cut-off value of ≥6 LCTCsmulti was also an effective prognosticator for poor median overall survival. Single cell-based DNA sequencing of 50 targeted CTCs indicated that SCTCstri and LCTCsmulti harbored distinct gene variations respectively. Mutations in the KRAS and Rap1 pathway were remarkably abundant in SCTCstri, whereas several unique mutations in the MET/PI3K/AKT pathway and SMARCB1 gene were identified in LCTCsmulti. Obtained results suggested that SCTCstri and LCTCsmulti exhibited different mechanisms to therapy resistance and correlated with patients' poor outcome.

Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer.

OBJECTIVE: Hematogenous metastasis is essential for the progression of ovarian cancer (OC), and circulating tumor cells (CTCs) are part of the metastatic cascade. However, the detection rate of CTC is low due to the use of less sensitive detection methods. Therefore, this study aimed to detect CTCs and circulating tumorigenic endothelial cells (CTECs) in patients with OC using subtraction enrichment and immunostaining and fluorescence in situ hybridization (SE-iFISH). METHODS: We enrolled a total of 56 subjects, including 20 OC patients and 36 ovarian benign tumor patients. CTCs and CTECs were captured by subtraction enrichment (SE) and counted and classified according to immunofluorescence staining of tumor markers (TMs) carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4) combined with fluorescence in situ hybridization (iFISH) of chromosome 8 (Chr8) aneuploidy. The diagnostic value and subtype characteristics of CTCs and CTECs were investigated. RESULTS: The detection rate of CTCs by SE-iFISH was high. Compared with CA125 and HE4, Chr8 aneuploidy was the major identification feature of CTC. CTC counts in OC were statistically higher than those in benign groups. CTC and CTEC with ≥pentaploidy were detected in both groups, illustrating the poor diagnostic value of CTC or CTEC. Distributions of triploid and tetraploid CTC subtypes were significantly different, and combined detection of triploid and tetraploid CTCs showed the best diagnostic value. In contrast, the distribution of CTECs in the OC and benign groups had no statistically significant difference. Small CTCs accounted for over 1/3 of the total CTC count. We also found that small CTCs and CTECs primarily comprised triploid cells, while large CTCs and CTECs mainly comprised pentaploidy and beyond. CONCLUSIONS: The application of SE-iFISH offered a more comprehensive understanding of heterogeneous CTCs and CTECs in OC. Analysis of subclass characteristics of the CTCs and CTECs according to Chr8 aneuploidy and cell size may broaden their potential clinical utility and deepen mechanistic studies in OC.

Small Cell Size Circulating Aneuploid Cells as a Biomarker of Prognosis in Resectable Non-Small Cell Lung Cancer.

OBJECTIVE: The size distribution of circulating aneuploid cells (CACs) and its clinical significance were investigated in resectable non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: A total of 50 patients with resectable NSCLC were enrolled in this study. Blood samples (50 pre-surgery and 35 post-surgery) were collected and used for the detection of CAC chromosome 8 heteroploidy through the subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) method. RESULTS: Less than 20% small cell size and more than 80% large cell size CACs were detected. Karyotypes, including triploid, tetraploid, and multiploid, had varying distributions. The triploid subtype accounted for the majority of small cell size CACs, whereas the multiploid subtype accounted for the majority of large cell size CACs. We found that total small cell size and triploid small cell size CACs, but not large cell size CACs, derived from pre-surgery samples, were associated with shorter disease-free survival. Moreover, total small cell size and triploid small cell size CACs were associated with higher TNM stage and recurrence. Nevertheless, the variation between pre- and post-surgery CACs was not related to survival among patients with resectable NSCLC. CONCLUSIONS: Pre-surgery small cell size CACs, especially the triploid subtype, could be regarded as a potential prognostic biomarker for patients with resectable NSCLC.

Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis.

Hematogenous and lymphogenous cancer metastases are significantly impacted by tumor neovascularization, which predominantly consists of blood vessel-relevant angiogenesis, vasculogenesis, vasculogenic mimicry, and lymphatic vessel-related lymphangiogenesis. Among the endothelial cells that make up the lining of tumor vasculature, a majority of them are tumor-derived endothelial cells (TECs), exhibiting cytogenetic abnormalities of aneuploid chromosomes. Aneuploid TECs are generated from "cancerization of stromal endothelial cells" and "endothelialization of carcinoma cells" in the hypoxic tumor microenvironment. Both processes crucially engage the hypoxia-triggered epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT). Compared to the cancerization process, endothelialization of cancer cells, which comprises the fusion of tumor cells with endothelial cells and transdifferentiation of cancer cells into TECs, is the dominant pathway. Tumor-derived endothelial cells, possessing the dual properties of cancerous malignancy and endothelial vascularization ability, are thus the endothelialized cancer cells. Circulating tumor-derived endothelial cells (CTECs) are TECs shed into the peripheral circulation. Aneuploid CD31+ CTECs, together with their counterpart CD31- circulating tumor cells (CTCs), constitute a unique pair of cellular circulating tumor biomarkers. This review discusses a proposed cascaded framework that focuses on the origins of TECs and CTECs in the hypoxic tumor microenvironment and their clinical implications for tumorigenesis, neovascularization, disease progression, and cancer metastasis. Aneuploid CTECs, harboring hybridized properties of malignancy, vascularization and motility, may serve as a unique target for developing a novel metastasis blockade cancer therapy.

Tumor biology and multidisciplinary strategies of oligometastasis in gastrointestinal cancers.

More than 70% of gastrointestinal (GI) cancers are diagnosed with metastases, leading to poor prognosis. For some cancer patients with limited sites of metastatic tumors, the term oligometastatic disease (OMD) has been coined as opposed to systemic polymetastasis (PMD) disease. Stephan Paget first described an organ-specific pattern of metastasis in 1889, now known as the "seed and soil" theory where distinct cancer types are found to metastasize to different tumor-specific sites. Our understanding of the biology of tumor metastasis and specifically the molecular mechanisms driving their formation are still limited, in particular, as it relates to the genesis of oligometastasis. In the following review, we discuss recent advances in general understanding of this metastatic behavior including the role of specific signaling pathways, various molecular features and biomarkers, as well as the interaction of carcinoma cells with their tissue microenvironments (both primary and metastatic niches). The unique features that underlie OMD provide potential targets for localized therapy. As it relates to clinical practice, OMD is emerging as treatable with surgical resection and/or other local therapy options. Strategies currently being applied in the clinical management of OMD will be discussed including surgical, radiation-based therapy, ablation procedures, and the results of emerging clinical trials involving immunotherapy.

PD-L1+ aneuploid circulating tumor endothelial cells (CTECs) exhibit resistance to the checkpoint blockade immunotherapy in advanced NSCLC patients.

Sustained angiogenesis and increased PD-L1 expression on endothelial and carcinoma cells contribute toward fostering an immunosuppressive microenvironment suitable for tumor growth. PD-L1+ CTCs were reported to associate with poor prognosis in NSCLC patients. However, whether or not aneuploid circulating tumor endothelial cells (CTECs) express PD-L1, then serve as a surrogate biomarker to evaluate immunotherapy efficacy remains unknown. In this study, a novel SE-iFISH strategy was established to comprehensively quantify and characterize a full spectrum of aneuploid CTCs and CTECs in advanced NSCLC patients subjected to second-line anti-PD-1 (nivolumab) immunotherapy. In situ co-detection of diverse subtypes of aneuploid CTCs and CTECs expressing PD-L1 and Vimentin was performed. The present clinical study demonstrated that significant amounts of PD-L1+ aneuploid CTCs and CTECs could be detected in histopathologic hPD-L1- patients. In contrast to decreased PD-L1+ CTCs, the number of multiploid PD-L1+ CTECs (≥tetrasomy 8) undergoing post-therapeutic karyotype shifting increased in patients along with tumor progression following anti-PD-1 treatment. Progressive disease (PD) lung cancer patients possessing multiploid PD-L1+ CTECs had a significantly shorter PFS compared to those without PD-L1+ CTECs. In carcinoma patients, aneuploid CTCs and CTECs may exhibit a functional interplay with respect to tumor angiogenesis, progression, metastasis, and response to immunotherapy.

Vimentin expression in circulating tumor cells (CTCs) associated with liver metastases predicts poor progression-free survival in patients with advanced lung cancer.

OBJECTIVE: To investigate the presence of vimentin expression in CTCs and its clinical relevance in patients with advanced lung cancer. METHODS: Peripheral blood was obtained from 61 treatment-naive patients with advanced lung cancer. Subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) platform was applied to identify, enumerate and characterize CTCs based on cell size, aneuploidy of chromosome 8 (Chr8) and vimentin expression. Quantification and analysis of CTCs were performed on patients before chemotherapy administration and after two cycles of therapy. RESULTS: Before treatment, CTCs were detected in 60 (98.4%) patients, small cell CTCs (≤ 5 µm of WBCs) accounted for 52.8% of the absolute CTCs number, while 12 (19.7%) of the included patients had detectable vimentin-positive CTCs (vim+ CTCs). Liver metastases were reported in 7 (11.5%) patients and were significantly correlated to the presence of Vim+ CTCs (p = 0.002), with a high positivity rate of 71.4% (5/7). Vim+ CTCs were mostly in small cell size and Chr8 aneuploidy (77.0% and 82.05%, respectively). Baseline small cell CTCs ≥ 2/6 ml, triploid CTCs ≥ 2/6 ml, Vim+ CTCs ≥ 1/6 ml were found to significantly correlate with poor progression-free survival (PFS) (p = 0.017, p = 0.009 and p = 0.001, respectively). After adjusting for clinically significant factors, baseline Vim+ CTCs ≥ 1/6 ml was the only independent predictor of poor PFS [hazard ratio (HR):2.756, 95% confidence interval (CI): 1.239-6.131; p = 0.013]. CONCLUSIONS: This study demonstrates an important morphologic, karyotypic and phenotypic CTCs heterogeneity in advanced lung cancer patients. The majority of Vim+ CTCs are in small size and Chr8 aneuploidy. Baseline presence of Vim+ CTCs is correlated with liver metastases and may help predict poor PFS.

Evolutionary Expression of HER2 Conferred by Chromosome Aneuploidy on Circulating Gastric Cancer Cells Contributes to Developing Targeted and Chemotherapeutic Resistance.

Purpose: Previous human epidermal growth factor receptor-2 (HER2)-derived resistance studies were based on ex vivo models, which could not mirror evolutionary expression of HER2 during therapy. To investigate dynamic expression of HER2 and its contribution to developing therapeutic resistance conferred by chromosome aneuploidy, both the HER2 phenotype and chromosome 8 (Chr 8) aneuploidy on circulating tumor cells (CTC) were coexamined in advanced gastric cancer (AGC) patients.Experimental Design: A total of 115 AGC patients, including 56 of histopathologic HER2+ (hHER2+) subjects who received first-line HER2-targeted therapy plus chemotherapy, and 59 of hHER2- patients who received chemotherapy alone, were prospectively enrolled. Both HER2 phenotype and Chr8 aneuploidy of CTCs in patients were coexamined by HER2-iFISH during therapy.Results: A fluctuated positive HER2 phenotype on CTCs (cHER2+) was revealed, showing cHER2+ at different time intervals during treatment. Acquisition of the cHER2+ phenotype in 91.0% of hHER2+ and 76.2% hHER2- patients was demonstrated to correlate with development of resistance to trastuzumab-targeted therapy for hHER2+ patients and chemotherapy alone for hHER2- patients. Aneuploid Chr8 was demonstrated to participate in the acquisition of the cHER2+ phenotype, which provides a growth advantage to HER2+ CTCs against therapeutic pressure, leading to the development of therapeutic resistance.Conclusions: Compared with low positivity of conventional histopathologic hHER2 examination routinely performed once, significant higher positivity of cHER2+ on CTCs was observed. Continuously examining cHER2 shows unique advantages with respect to monitoring therapeutic resistance in real time in carcinoma patients. Moreover, contribution of chromosome aneuploidy to the phenotypic evolution of HER2 expression on CTCs may help elucidate underlying mechanisms of developing therapeutic resistance. Clin Cancer Res; 24(21); 5261-71. ©2018 AACR.

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.

Clinical significance of detecting CSF-derived tumor cells in breast cancer patients with leptomeningeal metastasis.

Despite marked advances in breast cancer therapy, breast cancer-associated leptomeningeal metastasis (LM), a particularly aggressive syndrome with multifocal seeding of the leptomeninges by tumor cells, still carries an abysmal prognosis. A major problem with breast cancer LM surveillance is the lack of an effective and sensitive means to track dynamic changes of the disease. Cytology detection of cerebrospinal fluid (CSF) is considered the gold standard for LM diagnosis but has a high false-negative rate with a limited sensitivity. Here we applied subtraction enrichment and immunostaining-fluorescence in situ (SE-i•FISH) method, a technique previously used for isolating circulating tumor cells (CTCs) from the peripheral blood, to detect, enumerate, and track cerebrospinal fluid-derived tumor cells (CSFTCs) in CSF samples from 8 breast cancer patients. Comparing with cytology test, we found SE-i•FISH method can accurately and feasibly detect CSFTCs for the diagnosis of breast cancer-associated LM and monitor the disease progression. We also isolated and cultured CSFTCs from these cancer patients and performed genomic sequencing on CSFTCs of two patients. Genomic analysis of CSFTCs against corresponding archival primary breast tumors revealed clonal relationships with some ongoing evolution. Further drug sensitivity test on cultured CSFTCs based on genomic analysis data helped identify promising treatment options for the patient tested. Together, our results suggest that CSFTCs detection using SE-i•FISH platform could serve as a sensitive and accurate method to make the diagnosis and a promising approach to monitor tumor dynamics and treatment response for breast cancer-associated LM.