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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., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

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., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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 ( S CTCs tri ) or ≥6 large multiploid CTCs ( L CTCs multi ) showed an inferior median progression-free survival. Moreover, the cut-off value of ≥6 L CTCs multi was also an effective prognosticator for poor median overall survival. Single cell-based DNA sequencing of 50 targeted CTCs indicated that S CTCs tri and L CTCs multi harbored distinct gene variations respectively. Mutations in the KRAS and Rap1 pathway were remarkably abundant in S CTCs tri , whereas several unique mutations in the MET/PI3K/AKT pathway and SMARCB1 gene were identified in L CTCs multi . Obtained results suggested that S CTCs tri and L CTCs multi exhibited different mechanisms to therapy resistance and correlated with patients' poor outcome., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 ., (©2018 American Association for Cancer Research.)

Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45 - /CD31 - ) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM + aneuploid circulating tumor stem cells (CTSCs), and EpCAM - small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Conventional circulating tumor cell (CTC) detection strategies rely on cell surface marker EpCAM and intracellular cytokeratins (CKs) for isolation and identification, respectively. Application of such methods is considerably limited by inherent heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs. Here, we report a novel strategy, integrating antigen-independent subtraction enrichment and immunostaining-FISH (SE-iFISH), to detect a variety of aneuploid circulating rare cells (CRCs), including CTCs and circulating tumor endothelial cells (CECs). Enriched CRCs, maintained at high viability and suitable for primary tumor cell culture, are comprehensively characterized by in situ co-examination of chromosome aneuploidy by FISH and immunostaining of multiple biomarkers displayed in diverse fluorescence channels. We described and quantified for the first time the existence of individual aneuploid CD31 + CECs and co-existence of "fusion clusters" of endothelial-epithelial aneuploid tumor cells among enriched non-hematopoietic CRCs. Hence, SE-iFISH is feasible for efficient co-detection and in situ phenotypic and karyotypic characterization as well as quantification of various CRCs, allowing for their classification into diverse subtypes upon biomarker expression and chromosome ploidy. Enhanced SE-iFISH technology, assisted by the Metafer-iFISH automated CRC imaging system, provides a platform for the analysis of potential contributions of each subtype of CRCs to distinct clinical outcome.

Conventional strategy of anti-EpCAM capture and immunostaining of cytokeratins (CKs) to detect circulating tumor cells (CTCs) is limited by highly heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs. In this study, a novel integrated cellular and molecular approach of subtraction enrichment (SE) and immunostaining-FISH (iFISH) was successfully developed. Both large or small size CTCs and circulating tumor microemboli (CTM) in various biofluid samples including cerebrospinal fluid (CSF) of cancer patients and patient-derived-xenograft (PDX) mouse models were efficiently enriched and comprehensively identified and characterized by SE-iFISH. Non-hematopoietic CTCs with heteroploid chromosome 8 were detected in 87-92% of lung, esophageal and gastric cancer patients. Characterization of CTCs performed by CK18-iFISH showed that CK18, the dual epithelial marker and tumor biomarker, was strong positive in only 14% of lung and 24% of esophageal CTCs, respectively. Unlike conventional methodologies restricted only to the large and/or both EpCAM and CK positive CTCs, SE-iFISH enables efficient enrichment and performing in situ phenotypic and karyotypic identification and characterization of the highly heterogeneous CTC subtypes classified by both chromosome ploidy and the expression of various tumor biomarkers. Each CTC subtype may possess distinct clinical significance relative to tumor metastasis, relapse, therapeutic drug sensitivity or resistance, etc.

The HuPrime® human gastric neuroendocrine carcinoma derived xenograft model GA0087 was established in this study. GA0087 PDX model showed high gene expression of vascular endothelial growth factors (VEGF)-A and B, and high potential of lung metastasis. Circulating tumor cells (CTCs) with either large or small size, circulating tumor microemboli (CTM) and lung metastatic lesions were detected in GA0087 PDX mice. The number of CTC correlated to the number of metastatic nodules in lung. Both primary tumor growth and metastasis in terms of the number of dynamically monitored CTCs and metastatic nodules were effectively suppressed by Cisplatin. Diverse subtypes of CTCs in the context of sensitivity to Cisplatin were specifically identified by subtraction enrichment (SE) integrated with in situ Phenotyping of cytokeratin 18 (CK18) and Karyotyping of chromosome 8 (in situ PK CTC by CK-iFISH). All the CK18-/diploid and majority of CK18+/diploid CTC subtypes were chemosensitive, whereas a higher percentage of CK18+/multiploid subtype of CTC were Cisplatin-insensitive. Combined histopathological examination of metastatic lesion and in situ PK CTC in a metastatic PDX (mPDX) tumor model are of particular significance, and may provide an unique and robust platform for cancer research as well as pre-clinical evaluation of therapeutic efficacy of new anti-cancer drugs.

Background: Karyotyping and phenotyping of circulating tumor cells (CTCs) in therapeutic cancer patients is of particular clinical significance in terms of both identifying chemo-resistant CTC subtypes and understanding CTC evolution., Methods: The integrated subtraction enrichment (SET) and immunostaining-fluorescence in situ hybridization (iFISH) platform was applied to detect and characterize CTCs in patients with advanced gastric cancer (AGC). Status of human epidermal growth factor receptor 2 (HER2) expressing and aneuploidy of chromosome 8 in CTCs enriched from the patients was examined by SET-iFISH following clinical chemotherapy or HER2-targeted therapy. CellSearch system was applied as a reference control., Results: Phenotyping of CTCs in HER2 positive AGC patients demonstrated that HER2⁺ CTCs could be effectively eliminated in response to HER2-targeted therapy. Karytotyping of CTCs indicated that distinct CTCs with different ploidies of chromosomes 8 in AGC patients correlated to either sensitivity or resistance of paclitaxel or cisplatin-based chemotherapy. Examination of the copy number of chromosome 8 in CTCs provides a potential approach for predicting chemotherapeutic efficacy and monitoring chemo-resistance., Conclusions: Phenotyping and karyotyping of the enriched CTCs upon ploidy of chromosome 8 or HER2 expression is of clinical potential for monitoring chemo-resistance and evaluating therapeutic efficacy for AGC patients.