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

Keywords: EMT and EndoMT; EpCAM and vimentin; SE-iFISH; bevacizumab; prognosticators; therapy efficacy.

Fig. 1

Characteristics of the enrolled NSCLC patients. (A) Characteristics of patients and defined time intervals of CTC and CTEC assessment throughout combination therapy. The recruited 25 treatment‐naive advanced NSCLC adenocarcinoma (ADC) patients were subjected to first‐line combination regimen of platinum‐based chemotherapy and anti‐angiogenic bevacizumab for up to six cycles, followed by maintenance therapy composed of bevacizumab treatment. Detection of CTCs and CTECs was performed at the indicated time points of t ₀ (baseline), t ₁ (postcombination therapy, 2 cycles), and t ₂ (postcombination therapy, 4–6 cycles). (B) Quantitative illustration of patients and specimens throughout therapy. A total of 65 clinical samples in 25 patients were collected for assessment of overall CTCs and CTECs as well as their subtypes. A total of 21 patients are eligible for the follow‐up survival study.

Fig. 2

Quantification and molecular characterization of co‐detected diverse subtypes of aneuploid CTCs and CTECs. (A) Quantitative analysis of molecularly characterized CTCs and CTECs in different cell sizes. CTCs: among 659 CTCs, 220 of them are small cell sized _SCTCs (220 out of 659, 33.4%) with 20.6% (136 out of 659) being triploid (_SCTCs ^tri ); remaining 439 CTCs are large _LCTCs (439 out of 659, 66.6%) with 42.9% (283 out of 659) being multiploid (_LCTCs ^multi ). CTECs: out of 423 CTECs, 55 are _SCTECs (55 out of 423, 13%) with 4.7% (20 out of 423) being triploid (_SCTECs ^tri ); the rest of 368 cells are _LCTECs (368 out of 423, 87%) with 73.3% (310 out of 423) being multiploid (_LCTECs ^multi ). Highest percentages of different subtypes are indicated in red font. (B) Compositional waterfall map: compositions of CTC (Ba) and CTEC subtypes (Bb) are depicted in a schematic waterfall map. Percentages of each subtype as described in (A) are obtained from quantification analysis of total CTCs and CTECs longitudinally detected throughout therapy and indicated on the top of each column. (C) Representative images of CTC and CTEC subtypes identified by iFISH. (C‐a) A representative image of a large multiploid CTC (_LCTC ^multi ) expressing EpCAM (EpCAM⁺/vimentin (Vim)⁻/CD31⁻, epithelial E‐type). (C‐b) A representative image of a haploid mesenchymal small CTC (_SCTC ^mono ) with an EpCAM⁻/Vim⁺/CD31⁻ phenotype (mesenchymal M‐type). (C‐c) A representative image of a haploid small CTC (_SCTC ^mono ) expressing both EpCAM and vimentin (EpCAM⁺/Vim⁺/CD31⁻, intermediate hybrid E/M‐type). (C‐d) A representative image of a large multiploid E‐type CTEC (_LCTEC ^multi , EpCAM⁺/Vim⁻/CD31⁺). (C‐e) A representative image of a large multiploid M‐type CTEC (_LCTEC ^multi , EpCAM⁻/Vim⁺/CD31⁺). (C‐f) A representative image of a M‐type CTEC fusion cluster with multinuclei (EpCAM⁻/Vim⁺/CD31⁺) and a diploid CD45⁺ WBC attached (red arrow). (C‐g) A representative image of a large multiploid E/M‐type CTEC (_LCTEC ^multi , EpCAM⁺/Vim⁺/CD31⁺). All the representative images are from the image library of all patients’ CTCs and CTECs longitudinally detected throughout therapy as described in (A). Bars, 5 µm.

Fig. 3

Comprehensive analysis of heterogeneous‐sized CTCs and CTECs. (A) Categorization of patients. Based upon quantitative variation of CTCs (A‐a) and CTECs (A‐b) throughout therapy, patients are categorized into ascending (∆t ₂ red dot > ∆t ₁ blue dot) and descending (∆t ₂ < ∆t ₁) cohorts which are divided by a red dashed line. In each cohort, CTCs and CTECs exhibit a similar variation trend. (B) Heattable: longitudinal variation in cell numbers and percentages of the specific CTC and CTEC subtypes. Variation of the percentage of CTC or CTEC subtypes during treatment is similar to the cell number change in EpCAM⁺ or Vim⁺ CTCs and CTECs detected from t ₀ to t ₂. (C) Quantitative variation of CTCs and CTECs during therapy. (C‐a) CTC. In the ascending cohort (white bars), compared to the baseline median value (6 cells, t ₀), the post‐therapeutic median values of total CTCs exhibit a downward‐upward variation pattern of 3 cells at t ₁ and 12 cells at t ₂ (left y‐axis). Number of EpCAM⁺ CTCs (red) increased from 0 (t _0‐1) to 18 cells (t ₂) (right Y‐axis). In the descending cohort, the median values of total CTCs (gray bars, 5 cells at t ₀) display an upward‐downward variation pattern, showing 14 cells at t ₁ and 3 cells at t ₂, **P = 0.008 (t ₁ vs t ₂). EpCAM⁺ CTCs have the same upward‐downward pattern (red): 2 (t ₀), 13 (t ₁), and 0 cell (t ₂). Black dots: discrete data. (C‐b) CTEC. Total and EpCAM⁺ CTEC number in both ascending and descending cohorts respectively display similar downward‐upward and upward‐downward variation patterns. Differences in the median values of total CTECs are statistically significant, *P = 0.013 (t ₀ vs t ₂), and **P = 0.002 (t ₁ vs t ₂), log‐rank test. Quantitative changes in Vim⁺ CTCs or CTECs following therapy (blue) reveal an upward (t ₁)‐downward (t ₂) pattern in most cases.

Fig. 4

Prognosis analysis of the ascending and descending cohorts of patients. (A) Progressive clinical status of each patient in the ascending (red) and descending (blue) cohorts following therapy is illustrated. (B) Kaplan–Meier survival analysis. Cohorts classified by CTCs. The ascending cohort of patients shows a shortened median progression‐free survival (mPFS) of 3.0 months compared to the prolonged 9.4 months of the descending cohort (***P = 0.001, log‐rank test). Patients in the ascending cohort have a median overall survival (mOS) of 7.0 months, which is significantly shorter than 24.3 months of the descending cohort (***P = 0.001, log‐rank test). The ascending and descending cohorts categorized by CTECs have mPFS and mOS identical to that in cohorts classified by CTCs.

Fig. 5

Correlation of aneuploid CTC and CTEC subtypes with poor prognosis. (A) Sankey diagrams: longitudinal analysis of diverse subtypes of CTCs and CTECs detected at the indicated time intervals in a total of 21 patients during therapy. The number of subcategorized patients possessing each subtype of cells is indicated in the figure. (A‐a) CTC. There are three cohorts of patients respectively containing three diverse CTC subtypes at baseline (t ₀), including EpCAM⁻/vimentin (Vim)⁺ (M‐type, 3 patients), the most abundant EpCAM⁻/Vim⁻ (nonhematologic aneuploid N‐type null cells, 17 patients), and the least abundant EpCAM⁺/Vim⁺ (hybrid E/M‐type, 1 patient). Following therapy, a de novo cohort of patients acquiring EpCAM⁺/Vim⁻ (E‐type) CTCs emerges (red, t _1‐2, 7 patients), with most patients being the pretreatment cohort harboring N‐type null CTCs and a minority being the baseline M‐type patients (white arrows). Most patients hosting the new E‐type CTCs (red arrow) and the subject having the E/M‐type CTCs following therapy (black arrow) are toward poor prognosis (mPFS < 9.4 months). (A‐b) CTEC. Three cohorts of prior‐to‐therapy patients possess three distinct baseline CTEC subtypes, including M‐type (6 patients), N‐type null cell (9 patients), and E‐type CTECs (6 patients), are identified with relatively equal proportions at t ₀. Newly emerged hybrid E/M‐type CTECs (red, 5 patients) are detected in all three baseline cohorts during therapy (t _1‐2, white arrows). All five patients carrying the de novo E/M‐type (red arrow) and a majority of subjects possessing the E‐type post‐therapeutic CTECs (black arrow) exhibit an inferior prognosis. Most patients who had post‐therapeutic aneuploid N‐type null CTECs show a better response to treatment (green arrow, mPFS > 9.4 months). (B) Correlation of different cohorts of patients harboring diverse CTC and CTEC subtypes with disease progression. None of the baseline CTC subtypes (numbers in blue) are significantly relevant to poorer prognosis (PD with new mets) (P = 0.652), whereas post‐therapeutic EpCAM⁺ CTCs, regardless of Vim expression or not, significantly correlate with poorer prognosis (*P = 0.037, red). Both baseline Vim⁺ and post‐therapeutic EpCAM⁺ CTECs demonstrate a significant correlation with poorer prognosis, *P = 0.023 and *P = 0.017 (red), respectively. (C) Dichotomized Kaplan–Meier survival analysis. (C‐a) Pretherapeutic patients possessing baseline vimentin⁺ CTECs have a mPFS of 5.0 months compared with 10.1 months in those without vimentin⁺ CTECs (**P = 0.009, log‐rank test). (C‐b) Post‐therapeutic patients having EpCAM⁺ CTECs following therapy show a shorter mPFS of 5.6 months, whereas subjects who have no EpCAM⁺ CTECs reveal a prolonged mPFS of 18.3 months (**P = 0.002, log‐rank test). (C‐c) Compared to a mPFS of 10.1 months in post‐therapeutic patients without EpCAM⁺ CTCs, subjects with detectable EpCAM⁺ CTCs exhibited a reduced mPFS of 5.6 months (*P = 0.017, Breslow test).