Tumor-associated tertiary lymphoid structure predicts postoperative outcomes in patients with primary gastrointestinal stromal tumors

Abstract

Tumor-infiltrating tertiary lymphoid structures (TLS) are thought to have anti-tumor activity and are believed to indicate a favorable prognosis in cancer patients. However, the prognostic value of TLS in gastrointestinal stromal tumors (GIST) is unknown. We evaluated the prognostic value of TLS using two independent GIST cohorts. Pathological examinations identified TLS in 44.9% of patients in our discovery cohort (DC). TLS was significantly associated with smaller tumor size (P = .011), relatively well morphological classification (P < .001), lower NIH classification (P < .001), lower recurrence (P = .005), longer survival time (P < .001) and lower imatinib resistance (P = .006). Kaplan-Meier curves showed that TLS was remarkably associated with favorable survival (P = .0002) and recurrence (P = .0015) time. In addition, the presence of KIT mutations and the absence of TLS suggested worst prognosis both in terms of overall survival (OS) (P = .0029) and time to recurrence (TTR) (P = .0150), while the presence of PDGFRA mutations and TLS suggested optimal prognosis for OS and TTR. Multivariate analyzes demonstrated that TLS was an independent prognostic factor for OS (HR:0.180, P = .002) and TTR (HR:0.412, P = .023). These results were confirmed using our validation cohort. Multiplexed immunohistochemistry staining was used to determine the composition of TLS. Therapies designed to target TLS may be a novel therapeutic strategy for GIST patients with imatinib resistance.

Keywords: Tertiary lymphoid structure (TLS); gastrointestinal stromal tumor (GIST); imatinib; immune microenvironment; prognosis.

Figure 1.

The classification of tertiary lymphoid structure (TLS) in primary GIST. (a). Representative H&E image of TLS⁻ patients. Scale bar: 40 μm (left); 200 μm (right)(b). Representative H&E image of TLS Aggregates patients. (black dotted portion: TLS aggregates). Scale bar: 40 μm (left); 200 μm (right)(c). Representative H&E image of TLS Follicles patients. (black dotted portion: TLS Follicles; red dotted portion: germinal center). Scale bar: 40 μm (left); 200 μm (right)(d). Typical IHC staining of Ki67 in germinal center. (left: TLS aggregates; right, TLS Follicles). Scale bar: 400 μm.(e). TLS phenotype and frequency evaluated in discovery (left) and validation cohort (right). Proportions of TLS⁻, TLS Aggregates, Follicle I and Follicle II in GIST were summarized by pie chart.

Figure 2.

Immune profiles of TLS in GIST defined by multiplexed immunohistochemistry staining. (a). Representative 7-plex immunofluorescence image of T and B patterns of TLS follicles. Composite image (left) showing the colocalization staining pattern and the right panel shows a single-channel along. T cell panel 1 to show Treg cells (CD4⁺Foxp3⁺), Th17 cells (CD4⁺RORγt⁺), Th2 cells (CD4⁺GATA3⁺) and CD8⁺ T cells (CD8⁺). T cell panel 2 to show Trm cells (CD103⁺), Treg cells (CD4⁺Foxp3⁺), Th17 cells (CD4⁺RORγt⁺), Th2 cells (CD4⁺GATA3⁺) and Th1 cells (CD4⁺T-bet⁺). B cell panel to show plasma cells (PCs) (CD20⁻CD24⁻CD27^hiCD38^hi), B cells (CD20⁺), naive B cells (Bn) (CD20⁺CD27⁻IgM⁺), IgM⁺ memory B cells (IgM⁺ Bm) (CD20⁺CD27⁺IgM⁺), CD27⁻ isotype-switched memory B cells (CD27⁻ Sw Bm) (CD20⁺CD27⁻IgM⁻) and CD27⁺ isotype-switched memory B cells (CD27⁺ Sw Bm) (CD20⁺CD27⁺IgM⁻). Scale bars, 200 μm.(b). Representative images of five-color multiplex present immune profile in TLS⁺ patients, Scale bar: 200 μm.(c). Representative images of five-color multiplex present immune profile in TLS⁻ patients, Scale bar: 200 μm.

Figure 3.

Differences of TLS phenotype between imatinib resistant patients and non-imatinib resistant patients. (a). Representative H&E images in imatinib resistant (upper panel) and non-imatinib resistant patients (bottom panel). Scale bar: 40 μm (left); 200 μm (right).(b). Proportions of TLS⁻, Aggregates, Follicle I and Follicle II in our discovery cohort (upper panel) and validation cohort (bottom panel).(c). Kaplan-Meier curves of drug resistant time in patients with TLS⁺ and TLS⁻ (left, discovery cohort; right, validation cohort).

Figure 4.

Prognostic significance of TLS phenotypes of in GIST patients. (a). Kaplan-Meier analysis of overall survival (OS) and time to recurrence (TTR) in discovery cohort according to TLS phenotypes (n = 118).(b). Kaplan-Meier analysis of OS and TTR in validation cohort according to TLS phenotypes (n = 69).

Figure 5.

Prognostic significance of TLS phenotypes in GIST patients with the history of postoperative imatinib usage. (a). Kaplan-Meier analysis of overall survival (OS) and time to recurrence (TTR) in discovery cohort according to TLS phenotypes (n = 69).(b). Kaplan-Meier analysis of OS and TTR in validation cohort according to TLS phenotypes (n = 42).

Figure 6.

Association of mutation status with TLS phenotype in GIST patients. (a). Representative H&E images in KIT mutation patients. Scale bar: 40 μm (left), 200 μm (right).(b). Representative H&E images in PDGFRA mutation patients. Scale bar: 40 μm (left), 200 μm (right).(c). Representative H&E images in WT patients. Scale bar: 40 μm (left), 200 μm (right).(d). The percentages of TLS positive patients and TLS negative patients in different mutation status in discovery cohort and validation cohort.(e). Kaplan-Meier analysis of OS (left) and TTR (right) in patients with group III: KIT mutation and TLS⁻ (blue), group I: PDGFRA mutation and TLS⁺ (red), group II: others (green) in discovery cohort.(f). Kaplan-Meier analysis of OS (left) and TTR (right) in patients with group III: KIT mutation and TLS⁻ (blue), group I: PDGFRA mutation and TLS⁺ (red), group II: others (green) in validation cohort.