Disseminated tumor cells in bone marrow as predictive classifiers for small cell lung cancer patients

Abstract

Background: Small cell lung cancer (SCLC) is a highly aggressive disease characterized by early metastasis. Aneuploid CD31^- disseminated tumor cells (DTCs) and CD31⁺ disseminated tumor endothelial cells (DTECs) residing in the bone marrow are generally considered as the initiators of metastatic process. However, the clinical significance of DTCs and DTECs in SCLC remains poorly understood. The aim of this study is to investigate the clinical implications of diverse subtypes of highly heterogeneous DTCs and DTECs in SCLC patients.

Methods: Subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) was applied to enrich and perform comprehensive morphologic, karyotypic, and phenotypic characterization of aneuploid DTCs and DTECs in 30 patients. Additionally, co-detection of circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) was conducted on 24 of the enrolled patients. Proof-of-concept of the whole exon sequencings (WES) on precisely selected different subtypes of CTCs or DTCs, longitudinally detected from a representative case with pathologically confirmed bone marrow metastasis, was validated to feasibly reveal genetic mutations in these cells.

Results: DTCs, DTECs and their subtypes were readily detectable in SCLC patients. Comparative analysis revealed that the number of DTCs and DTECs was significantly higher than that of their corresponding CTCs and CTECs (P < 0.001 for both). Positive detection of disseminated tumor microemboli (DTM) or disseminated tumor endothelial microemboli (DTEM) was associated with inferior survival outcomes (P = 0.046 and P = 0.048). Patients with EpCAM⁺ DTCs detectable displayed significantly lower disease control rate (DCR) (16.67% vs 73.33%, P = 0.019), reduced median progression-free survival (mPFS) and median overall survival (mOS) compared with those with EpCAM^- DTCs (P = 0.028 and P = 0.002, respectively). WES analysis indicated that post-treatment DTCs isolated from bone marrow at the time of disease progression shared more homologous somatic gene mutations with pre-treatment CTCs compared with post-treatment CTCs.

Conclusions: Our findings suggest that bone marrow sampling and characterization of DTC subtypes provided a valuable tool for predicting treatment response and the prognosis in SCLC. Moreover, DTCs inherit a greater amount of homologous somatic information from pre-treatment CTCs, indicating their potential role in disease progression and treatment resistance.

Keywords: Aneuploid DTCs and DTECs; Bone marrow; Prognosis; SCLC; SE-iFISH.

Fig. 1

The overall design of this study. Created with BioRender.com. CEP8, centromere probe 8; CNV, copy number variation; CTCs, circulating tumor cells; CTECs, circulating tumor endothelial cells; DAPI, 4,6-diamino-2-phenyl indole; DNA, deoxyribo nucleic acid; DTCs, disseminated tumor cells; DTECs, disseminated tumor endothelial cells; DTEM, disseminated tumor endothelial microemboli; DTM, disseminated tumor microemboli; Epcam, Epithelial cell adhesion molecule; InDel, insertion deletion; SE-iFISH, subtraction enrichment and immunostaining-fluorescence in situ hybridization; SNV, single nucleotide variation; WES, whole exon sequencing.

Fig. 2

Representative images of heterogeneous aneuploid DTCs and DTECs expressing EpCAM or Vim in SCLC patients by SE-iFISH. (A) A representative image of a large multiploid (≥ pentasomy 8) EpCAM⁺/Vim^-/CD31^- DTCs (_LDTC^multi). (B) A representative image of a small EpCAM^-/Vim⁺/CD31^- triploid DTCs (_SDTC^tri). (C) A representative image of a small EpCAM^-/Vim^-/CD31^- triploid DTCs. (D) A representative image of a large multiploid DTECs with phenotypes of EpCAM⁺/Vim^-/CD31⁺ (_LDTEC^multi). (E) A representative image of a small triploid EpCAM^-/Vim^-/CD31⁺ DTEC. (F) A representative image of an EpCAM⁺/Vim^-/CD31^- fusogenic disseminated tumor microemboli. CEP8, centromere probe 8; DAPI, 4,6-diamino-2-phenyl indole. DTCs, disseminated tumor cells; DTECs, disseminated tumor endothelial cells; DTEM, disseminated tumor endothelial microemboli; DTM, disseminated tumor microemboli; Epcam, epithelial cell adhesion molecule; SCLC, small cell lung cancer; SE-iFISH, subtraction enrichment and immunostaining-fluorescence in situ hybridization; Vim, vimentin.

Fig. 3

Quantification and molecular characterization of co-detected diverse subtypes of aneuploid disseminated rare cells (DTCs + DTECs) and circulating rare cells (CTCs + CTECs). (A-a) Quantitive distribution of pre-treatment DTCs and DTECs. (A-b) Quantitive distribution of pre-treatment CTCs and CTECs. (B-a) Shown are numbers comparison of detected DTCs and CTCs in each patient. Paired DTC and CTC values are connected by solid lines (P < 0.001, Wilcoxon signed-rank test). (B-b) Shown are numbers comparison of detected DTECs and CTECs in each patient. Paired DTEC and CTEC values are connected by solid lines (P < 0.001, Wilcoxon signed-rank test). (C) Quantitative analysis of molecularly characterized DTCs and DTECs with different phenotypes. Highest proportions of different subtypes are highlighted in red font. (D) Compositions of DTC (D-a) and DTEC (D-b) subtypes are depicted in a waterfall map. CTCs, circulating tumor cells; CTECs, circulating tumor endothelial cells; DTCs, disseminated tumor cells; DTECs, disseminated tumor endothelial cells.

Fig. 4

Kaplan–Meier survival analysis of DTM and DTEM on SCLC patients' OS. (A) The OS curves of SCLC patients with negative and positive DTM. (B) The OS curves of SCLC patients with negative and positive DTEM. DTM, disseminated tumor microemboli; DTEM, disseminated tumor endothelial microemboli; OS, overall survival; SCLC, small cell lung cancer.

Fig. 5

WES of CTCs and DTCs from an extensive disease small cell lung cancer patient with pathologically confirmed bone marrow metastasis at the time of tumor progression. (A) Gene mutation distribution of the three samples is graphically depicted by the log value and summarized in the heatmap. (B) Venn diagram showed distribution of the overlap SNV mutations between each sample. (C) Pan-cancer mutation genes are listed as overlapped mutation genes (blue) and sample specific mutated genes (red). CTCs, circulating tumor cells; DTCs, disseminated tumor cells; WES, whole exon sequencing.

Fig. 6

The relationship between EpCAM⁺ DTCs, EpCAM⁺ DTECs, and DCR in SCLC. (A) Response after two cycles of treatment administration in all enrolled SCLC patients. (B) Patients with positive detection of EpCAM⁺ DTCs were significantly correlated with a decreased DCR (P = 0.019), but no statistical difference in DCR was observed between EpCAM⁺DTECs and EpCAM^- DTECs cohort patients (P = 0.449). DCR, disease control rate; DTC, disseminated tumor cells; DTEC, disseminated tumor endothelial cells; Neg, negative; PD, progressive disease; Pos, positive; PR, partial response; SD, stable disease; SCLC, small cell lung cancer.

Fig. 7

Prognosis analysis of the EpCAM⁺ DTCs and EpCAM^- DTCs cohorts of patients. (A) Progressive clinical status of each subject in the EpCAM⁺ DTCs (red) and EpCAM^- DTCs (blue) cohorts is illustrated. (B) Kaplan–Meier survival analysis. (Ba) The PFS curves of overall SCLC patients with EpCAM⁺ DTCs and EpCAM^- DTCs. (Bb-c) The PFS curves of patients with EpCAM⁺ DTCs and EpCAM^- DTCs in limited-stage SCLC and extensive-stage SCLC cohorts. (Bd) The OS curves of overall SCLC patients with EpCAM⁺ DTCs and EpCAM^- DTCs. (Be-f) The OS curves of patients with EpCAM⁺ DTCs and EpCAM^- DTCs in limited-stage SCLC and extensive-stage SCLC cohorts. DTCs, disseminated tumor cells; Epcam, epithelial cell adhesion molecule; mo, months; mOS, median overall survival; PD, progressive disease; PFS, progression-free survival, Pos, positive; PR, partial response; SCLC, small cell lung cancer; SD, stable disease.