ABSTRACT
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.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Neoplastic Cells, Circulating , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Neoplastic Cells, Circulating/pathology , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Longitudinal Studies , Tetraploidy , Prognosis , Disease Progression , Biomarkers, Tumor/geneticsABSTRACT
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.
Subject(s)
Glioma , Neoplastic Cells, Circulating , Aneuploidy , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Drug Resistance, Neoplasm , Endothelial Cells/metabolism , ErbB Receptors , Glial Fibrillary Acidic Protein , Glioma/genetics , Humans , In Situ Hybridization, Fluorescence , Neoplastic Cells, Circulating/pathology , PrognosisABSTRACT
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.
Subject(s)
Gastrointestinal Neoplasms/etiology , Gastrointestinal Neoplasms/metabolism , Biomarkers , Combined Modality Therapy , Disease Management , Disease Progression , Disease Susceptibility , Gastrointestinal Neoplasms/pathology , Gastrointestinal Neoplasms/therapy , Humans , Karyotype , Neoplasm Metastasis , Phenotype , Treatment OutcomeABSTRACT
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.
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OBJECTIVE: Previous work indicated that aneuploidy of chromosome 8 in circulating tumor cells (CTCs) correlated with therapeutic efficacy for advanced gastric cancer (AGC) patients. In this follow-up study performed on the same population of AGC patients, we investigated whether and how aneuploidy of chromosome 8 in CTCs correlates with patients' clinical prognosis. METHODS: The prospective study was performed on 31 patients with newly diagnosed AGC. Previously established integrated subtraction enrichment (SE) and immunostaining-fluorescence in situ hybridization (iFISH) platform was applied to identify, enumerate and characterize CTCs. Quantification of CTCs and analysis of their aneuploidy of chromosome 8 were performed on patients before and after therapy. RESULTS: CTCs were measured in 93.5% of AGC patients, and two CTC subtypes with diverse threshold values were identified, multiploid CTCs with the threshold of ≥2 per 7.5 mL and multiploid plus triploid CTCs with the threshold of ≥4, which were found to significantly correlate with poor progression-free survival (PFS) and overall survival (OS). In particular, patients with ≥10% increased multiploid CTCs after an initial 6 weeks of therapy had poor PFS and OS, whereas improved PFS and OS were observed on those who had ≥10% decreased multiploid CTCs. After adjusting for clinically significant factors, ≥10% increased post-therapy multiploid CTCs was the only independent predictor of PFS and OS. CONCLUSIONS: Aneuploidy of CTCs correlates with prognosis of AGC patients. Quantitative comparison monitoring multiploid CTCs before and after therapy may help predict improved or inferior prognosis and chemoresistance.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Aneuploidy , Endothelial Cells , Margins of Excision , Melanoma , Humans , Melanoma/pathology , Melanoma/immunology , Melanoma/genetics , Melanoma/therapy , Endothelial Cells/pathology , Endothelial Cells/metabolism , Immunotherapy/methods , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Neoplastic Cells, Circulating/pathology , Neoplastic Cells, Circulating/metabolism , In Situ Hybridization, Fluorescence , Male , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Skin Neoplasms/pathology , Skin Neoplasms/immunology , Skin Neoplasms/genetics , Skin Neoplasms/therapy , Middle Aged , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/genetics , FemaleABSTRACT
A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , ErbB Receptors/genetics , Lung Neoplasms/genetics , Mutation , Neoplastic Cells, Circulating/metabolism , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/pathology , ErbB Receptors/blood , Exons , Genome, Human , Humans , Laser Capture Microdissection , Lung Neoplasms/blood , Lung Neoplasms/diagnosis , Lung Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Nucleic Acid Amplification Techniques , Sequence Analysis, DNA , Single-Cell AnalysisABSTRACT
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.
Subject(s)
Lung Neoplasms , Neoplastic Cells, Circulating , Small Cell Lung Carcinoma , Humans , Small Cell Lung Carcinoma/drug therapy , Prognosis , Neoplastic Cells, Circulating/pathology , Prospective Studies , Endothelial Cells/pathology , Biomarkers, Tumor/genetics , Aneuploidy , Lung Neoplasms/pathologyABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
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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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Neoplastic Cells, Circulating , Aneuploidy , Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung/pathology , Endothelial Cells/metabolism , Humans , Lung Neoplasms/pathology , Neoplastic Cells, Circulating/pathologyABSTRACT
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.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Drug Resistance, Neoplasm/genetics , Neoplastic Cells, Circulating/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Aneuploidy , Chromosomes, Human, Pair 8/genetics , Female , Genomics/methods , Humans , Male , Middle Aged , Mutation/genetics , Progression-Free Survival , Signal Transduction/genetics , Trisomy/geneticsABSTRACT
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.
Subject(s)
Aneuploidy , Endothelial Cells/metabolism , Neoplastic Cells, Circulating/metabolism , Neovascularization, Pathologic/metabolism , Humans , Neoplasm MetastasisABSTRACT
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.
Subject(s)
B7-H1 Antigen/genetics , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Neoplastic Cells, Circulating/immunology , Neovascularization, Pathologic/drug therapy , Aneuploidy , B7-H1 Antigen/antagonists & inhibitors , Biomarkers, Tumor/immunology , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Disease Progression , Drug Resistance, Neoplasm/immunology , Endothelial Cells/immunology , Female , Humans , Immunotherapy , Male , Middle Aged , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/pathology , Nivolumab/administration & dosage , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/genetics , Progression-Free Survival , Tumor Microenvironment/immunologyABSTRACT
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.
Subject(s)
Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Neoplastic Cells, Circulating/metabolism , Vimentin/metabolism , Chromosomes, Human, Pair 8/genetics , Female , Follow-Up Studies , Humans , Male , Middle Aged , Progression-Free Survival , Prospective StudiesABSTRACT
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.