June 22, 2024

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Advances in immunotherapy for gastrointestinal stromal tumors

Advances in immunotherapy for gastrointestinal stromal tumors



Advances in immunotherapy for gastrointestinal stromal tumors.
Mesenchymal gastrointestinal cancer, typified by gastrointestinal stromal tumor ( GIST ), occurs throughout the gastrointestinal tract and affects human health and the economy globally.

The global annual incidence of GIST is about 10-15 per million, and the incidence is much higher in East Asia compared with North America.

About 20-30% of GIST patients are malignant, and the five-year survival rate of malignant GIST patients is about 35-65%, which seriously threatens human health.

Advances in immunotherapy for gastrointestinal stromal tumors

Primary GISTs can occur anywhere throughout the GI tract, but most originate in the stomach ( 60% ) and small intestine ( 30% ).

Metastasis is common in advanced GIST, usually to the liver ( 50-65% ) and peritoneum ( 20-43% ), and other sites are relatively rare.

GIST is widely believed to originate from interstitial cells of Cajal ( ICCs ), the pacemaker cells lining the gastrointestinal wall, and is mainly caused by mutations in driver genes.

Mutations in KIT and platelet-derived growth factor receptor α ( PDGFRA ) cause constitutive activation of KIT and PDGFRA signaling pathways in a ligand-independent manner, respectively, which is the main molecular mechanism for the occurrence and development of GIST.

Both KIT and PDGFRA are homologous type III receptor tyrosine kinases.

According to the mutation of the driver gene, GIST can be roughly divided into KIT mutant GIST, PDGFRA mutant GIST, and KIT/PDGFRA wild-type ( WT ) GIST.

Over the past two decades, the discovery of imatinib and other tyrosine kinase inhibitors ( TKIs ) has revolutionized the treatment of recurrent/metastatic or unresectable GIST.

However, TKIs often have transient disease control and very limited efficacy due to primary and secondary resistance.

Therefore, new therapeutic targets and drugs are urgently needed to circumvent drug resistance and further improve the therapeutic effect.


The immune landscape of GIST

Several studies explored the immune landscape of GIST and found that almost all GIST samples were infiltrated by varying numbers of immune cells. Macrophages and T lymphocytes are the most common immune cells in GIST.

In addition, some less abundant immune cells were infiltrated in GIST, including natural killer ( NK ) cells, B cells, dendritic cells ( DC ), natural killer T ( NKT ) cells, γδT cells, neutrophils, Eosinophils and mast cells, etc.

The intratumoral distribution pattern of immune cells in GIST is variable; they are predominantly diffusely distributed around blood vessels and less commonly distributed in aggregates.

Infiltrating immune cells in GIST have been shown to be associated with patients’ clinicopathological features and have predictive value.

tumor-associated macrophages

Tumor-associated macrophages ( TAMs ) are one of the most common inflammatory cells in the GIST microenvironment and can be divided into M1 and M2 macrophages.

M1 macrophages exert antitumor activity by phagocytizing tumor cells, presenting tumor cell antigens to T cells, and producing proinflammatory cytokines; in contrast, M2 macrophages promote tumor progression by suppressing inflammatory responses, recruiting Treg cells, and stimulating angiogenesis .

In untreated primary GIST, the polarization of TAMs in the microenvironment is controversial, and either M2 or M1 macrophages may be the predominant cell subtype.

Whereas in metastatic and imatinib-treated GIST, M2 macrophages were the most enriched subtype with high levels of expression of major histocompatibility complex class II (MHC-II) molecules .

Tumor infiltrating T lymphocytes

T lymphocytes in GIST include CD8+T, CD4+T and a small amount of Foxp3+T regulatory (Treg) cells. CD8+ T lymphocytes are the key lymphocytes to kill cancer cells and generate therapeutic responses to anti-PD-1 antibodies.

GIST has been found to have high infiltrating levels of CD8+ T cells, and CD8+ T lymphocytes in GIST have been shown to be positively correlated with RFS and OS. As for CD4+ T cells, they also infiltrated almost all GIST samples, but the numbers were not as abundant as CD8+ T cells.

So far, the prognostic value of CD4+ T cell infiltration in GIST has not been well evaluated.

Treg cells that suppress antitumor immune responses are relatively rare in GIST.

Treg cell infiltration in GIST was negatively associated with PFS but positively associated with high-risk GIST, and no association between Treg cell infiltration and GIST metastasis was found.

Furthermore, the infiltration of Treg cells in GIST was positively correlated with that of M2 macrophages, suggesting that the immunosuppressive effect of the latter might be attributed to the recruitment of Treg cells.

The ratio of CD8+T/Foxp3+Treg cells in GIST is much lower than that in cervical cancer, which indicates that there is strong immunosuppression in the GIST microenvironment.

Tumor infiltrating NK cells

NK cells belong to the innate immune system and are the first-line defense system against infection and tumors.

They can target cells with low MHC-I expression and serve as an important complement to cell-mediated anti-tumor immunity.

Unlike other solid tumors, NK cells are abundant in gastrointestinal stromal tumors, about 42.1% of GISTs have CD56+ NK cells, and about 25% of CD45+ cells are CD3-CD56+ NK cells.

NK cells infiltrating in GIST were positively correlated with PFS, RFS, OS, and prognosis, and negatively correlated with high-risk GIST, proliferation index, and metastasis.

NK cells express a variety of receptors, such as NKp30 and NKp46, which endow NK cells with different functions.

In GIST, NK cells mainly express the immunosuppressive NKp30C isoform, and its expression level is negatively correlated with OS and prognosis. B7-H6, a ligand of NKp30, is ubiquitously expressed in GIST; its soluble form, sB7-H6, is inversely associated with DFS and prognosis in metastatic GIST.

Furthermore, the presence of TGF-β in Treg cells downregulated the expression of NKG2D receptors in NK cells, which directly suppressed the latter’s cytotoxicity.

Tumor-infiltrating B cells and tertiary lymphoid structures

Tumor-infiltrating B cells in tertiary lymph nodes are critical in antitumor immune responses; they promote humoral antitumor responses through antibody-dependent cellular cytotoxicity ( ADCC ) and complement-dependent cytotoxicity ( CDC ).

In GIST, B lymphocytes were negatively correlated with tumor size and positively correlated with RFS and OS.

Tertiary lymphoid structures ( TLSs ), which typically include T-cell and B-cell follicular zones, are ectopic lymphoid aggregates that are widely present in various cancer types.

Tumor-infiltrating TLSs were found in 44.9%-52.2% of GISTs, whose outer layer was mainly composed of CD4+ T and CD8+ T cells, while the inner layer was mainly composed of B cells.

TLSs were found to be associated with lower-risk GIST, longer survival time, RFS, and lower imatinib resistance, and thus may be a new therapeutic strategy for patients with imatinib-resistant GIST.

Immune Cell Infiltration and Metastasis

The level of immune cell infiltration was closely related to the metastatic status of GIST . In primary GIST , the number of CD68+ macrophages was higher than that of CD3+ T cells, while the opposite was true for metastatic GIST .

Metastatic GIST contained approximately twice as many M2 macrophages as primary GIST , suggesting that M2 macrophages contribute to tumor progression.

In addition, the numbers of infiltrating CD3+ T , B , and NK cells in metastatic GIST were also much higher than those in primary GIST .

At the tumor margin, local non-metastatic GIST exhibited more CD8+ T and Foxp3+ Treg cells than metastatic GIST .

In addition, the infiltration level of immune cells was also closely related to the metastatic site of GIST . Liver metastases have moreCD3+T , CD56+NK and CD20+B cells.

Advances in immunotherapy for gastrointestinal stromal tumors


Immune checkpoints and other immune-related genes

PD1/PD-L1

In GIST, PD-L1 mainly exists on tumor cells, and a small amount exists in white blood cells, regardless of the mutation type of the driver gene. Its expression shows high heterogeneity in GIST; about 16.3%-69.0% of GIST samples have PD-L1 expression, and in the same sample, less than 10% of tumor cells may express PD-L1. Overall, the expression level of PD-L1 was relatively low in GIST.

PD-1 is mainly expressed at low levels on GIST-infiltrating T cells. PD-1 was reported to be present in 48.5% or 88% of GIST samples. PD-1 expression was higher in intratumoral T cells than in circulating T cells, and its expression was not associated with RFS or OS.

IDOs

Indoleamine 2,3-dioxygenase ( IDO ) is the rate-limiting enzyme in human tryptophan metabolism, which metabolizes tryptophan to kynurin.

IDO plays an immunosuppressive role by directly inhibiting the activity of CD8+ T cells and inducing the differentiation of Treg cells.

Furthermore, tryptophan metabolites are able to polarize antigen-presenting cells ( APCs ), exhibiting an immune tolerance phenotype characterized by the secretion of TGF-β or IL-10.

In GIST, constitutively activated KIT signaling upregulates IDO expression through the transcription factor ETS variant 4 ( ETV4 ).

The expression level of IDO in GIST is high, 63%-89.8% of GISTs are IDO-positive, and almost all PD-L1+GISTs are IDO+GIST. Furthermore, all PDGFRA mutant GISTs expressed IDO.

Tim-3/Gal-9 and other immune checkpoints

In GIST, Tim-3 is mainly present in TILs with a low expression level. Galectin-9 (Gal-9), the ligand of Tim-3, is mainly expressed in tumor cells. Almost all Tim-3+NK-infiltrated GIST samples showed Gal-9 expression, suggesting that their interaction may be involved in the suppression of antitumor immunity.

The expression level of Tim-3 in GIST was positively correlated with OS, PFS and CD8+ T cell density, and negatively correlated with high-risk GIST. In contrast to Tim-3, the expression of Gal-9 was positively correlated with high-risk GIST, negatively correlated with the density of CD8+ T cells and CD56+ NK cells, and not correlated with OS and PFS. These conflicting findings suggest that Tim-3 and Gal-9 may have different immune escape mechanisms in GISTs.

LAG-3 and VISTA are mainly expressed on the surface of T cells. LAG-3 expression was low in GIST, and VISTA expression in GIST was associated with improved outcome.

Furthermore, ICOS and its ligand B7H2 ( ICOSL ) are also present in GIST and are associated with poor prognosis. In addition, B7-H6 is highly expressed in GIST and may become a potential immunotherapy target.

CTA, MHC-I and MICA/B

Cancer-testis antigens ( CTAs ) are a large class of tumor-associated antigens, and CTAs are expressed in various malignant tumors, while in normal tissues, CTAs are only present in the germ cells of the testis and placenta; Ideal target for immunotherapy. It has been reported that 26.7%-40% of GIST patients exhibit CTAs expression.

MHC molecules, also known as human leukocyte antigens ( HLAs ), are mainly expressed on the surface of tumor cells and antigen-presenting cells.

However, only 30% of GIST samples showed normal expression levels of MHC-I molecules, and most of these samples had partial defects in HLA expression; 38% of GISTs had no expression of HLA-A, and 20% had no expression of HLA-B and HLA-C .

Defective expression of MHC-I molecules leads to reduced recognition of tumor cells by cytotoxic T lymphocytes and impairs antitumor immune responses.

Although MHC-I expression was lost in GIST, MHC class I chain-associated proteins A and B ( MICA/B ) were found in GIST , suggesting that NK cells may play a key role in the antitumor immune response.

Other immune-related genes

By releasing a variety of molecules, tumor cells are able to recruit different types of cells into the tumor tissue, alter the tumor microenvironment, and in turn promote its growth and metastasis.

The expression of inflammatory cytokines in GIST was very low, and the expression of TNFα was basically negligible, indicating the presence of an immunosuppressive microenvironment in GIST .

As for chemokines, such as CCL2 , CCL3 , and CXCL1 , their expression is relatively high in GIST , and CCL2 induces macrophage infiltration into tumor tissue and promotes tumor growth.

In addition, the GIST microenvironment contains a large amount of TGF-β1 , which can reduce the activity of immune effector cells and promote tumor metastasis.


Immunity therapy

Due to the large number of immune cell infiltration in GIST, immune cells and molecules are considered to play an important role in the occurrence and development of GIST. Therefore, therapeutics targeting the immune microenvironment of GIST are considered to reactivate the anti-tumor immunity of the host immune system and improve the therapeutic effect of imatinib, which may become a new strategy to solve the bottleneck of GIST treatment in the future. So far, several immunotherapies have been reported, including cytokine therapy, ICIs, antibody therapy, antibody drug conjugates ( ADC ), tumor vaccines, and adoptive cell therapy ( ACT )

Advances in immunotherapy for gastrointestinal stromal tumors

Cytokine-based immunotherapy

Type I IFNs contain IFNα and IFNβ and are mainly produced by macrophages, DCs and neutrophils infiltrating the tumor microenvironment. The long-acting interferon-α2b ( PegIFNα2b ) formed by the combination of polyethylene glycol ( PEG ) and recombinant interferon-α-2b has been used to treat GIST. In a study of eight patients with stage III/IV GIST taking PegIFNα2b, it was found that 4 weeks of combined treatment induced a large amount of IFN-γ and increased the production of IFN-γ-producing CD4+ T, CD8+ T and NK cells. infiltration. After a median follow-up of 3.6 years, the total effective rate reached 100%, and combination therapy was superior to imatinib alone. Further studies showed that the combination therapy has a synergistic effect and reversal of imatinib resistance.

immune checkpoint inhibitors

Immune checkpoint inhibitors ( ICIs ) are the most common, effective, and promising immunotherapy drugs, including anti-PD-1 antibodies, anti-CTLA-4 antibodies, and anti-PD-L1 antibodies. Although ICIs have not been approved for the treatment of GIST, many clinical trials are underway. Unfortunately, however, most clinical trials have been largely unsuccessful; few significant synergies have been found between TKIs and ICIs.

However, we cannot conclude that ICIs are ineffective because a small number of patients with advanced GIST achieved stable disease ( SD ) or partial response ( PR ) with ICIs and combined therapy. There are currently no conclusive biomarkers available for prediction and selection of GIST patients who may benefit from ICI immunotherapy.

Antibody-Based Immunotherapy

Monoclonal antibodies have been widely used to treat various types of tumors. A number of studies have evaluated the antitumor effects of antibodies against KIT and CD40 in mouse GIST models, as well as antibodies against PDGFRA, VEGF, and XmAb18087 ( a bispecific antibody targeting both SSTR2 and CD3 ) in GIST patients.

A series of studies have shown that the anti-KIT monoclonal antibody SR1 reduces the expression of KIT in tumor cells, enhances the phagocytosis of macrophages, and induces tumor cell death; its killing effect is related to the sensitivity or resistance of imatinib Sex has nothing to do. In addition, in the KitV558Δ/+ mouse GIST model, anti-CD40 antibody activated tumor-associated macrophages to produce TNF-α and enhanced the antitumor activity of imatinib.

In GIST, VEGF expression is associated with poor prognosis. Studies have evaluated the anti-VEGF monoclonal antibody bevacizumab in patients with metastatic/unresectable GIST; however, due to the small number of GIST patients enrolled and the unsatisfactory efficacy of bevacizumab, clinical trials Termination, no comprehensive conclusions were drawn. In 2017, the anti-PDGFRA monoclonal antibody olaratumab was clinically studied in 31 patients with metastatic and/or unresectable GIST; no CR and PR were observed, and only 5 patients had SD. XmAb18087, is a bispecific antibody targeting both somatostatin receptor 2 ( SSTR2 ) and CD3, the former being highly expressed in GIST. A clinical trial ( NCT03411915 ) is currently underway investigating the treatment in patients with advanced GIST and neuroendocrine tumors.

Antibody Drug Conjugate

So far, two anti-KIT ADCs ( LOP628-DM1 and NN2101-DM1 ) and one anti-GPR20 ADC ( DS-6157a ) have been developed and tested in GIST . LOP628-DM1 is sensitive in tumor cells with high KIT levels, suggesting that this ADC can be used to treat KIT mutant and KIT-WT GIST, but hypersensitivity reactions (HSRs) caused by LOP628-DM1 may limit its clinical application use. Likewise, NN2101-DM1 was also found to inhibit tumor growth in GIST in vivo and in vitro. DS-6157a, which is a drug conjugate of anti-GPR20 antibody and Dxd, is currently undergoing a phase I clinical trial to test the anti-tumor effect of DS-6157a in GIST patients.

Vaccine-Based Immunotherapy

Vaccines are a new approach to tumor immunotherapy. Ilixadensel, also known as Intuvax, is an allogeneic DC vaccine that elicits an antitumor immune response after intratumoral injection. The efficacy of ilixadencel has been evaluated in 6 patients with unresectable or metastatic GIST, and 33% of patients responded, supporting the need for further studies in the future.

Adoptive cell therapy

So far, three research teams have explored the efficacy of ACT in GIST. The first to construct KIT-targeting chimeric antigen receptor T ( CAR-T ) cells in 2013, and then demonstrated in a CDX model that these cells were able to produce IFN-γ in vitro, lyse cultured GIST cells and inhibit tumor growth . In the second study in 2019, eight patients with refractory recurrent GIST received intravenous allogeneic phytohemagglutinin ( PHA )-activated T cells, a personalized vaccine, and low-dose interleukin-2, five of whom were in Remission occurred after 14 months of follow-up study, with 1 SD and 2 PD, showing that this treatment is safe and effective. The third obtained cytokine-induced killer cells ( CIKs ) from KIT/PDGFRA-WT GIST patients in 2022 and found that patient-derived CIKs could directly or indirectly kill autologous imatinib and sunitinib drug-resistant tumor cells.


Summary

The tumor microenvironment of GIST is severely immunosuppressive, manifested by infiltration of M2 macrophages and Treg cells, high expression of IDO on GIST cells, immunosuppressive receptors on NK cells, and insufficient expression of MHC-I on APCs .

Although CD8+ T cells are enriched in GIST , the CD8+ T/Treg cell ratio is relatively low and may be further suppressed by PD-L1 on GIST cells .

Furthermore, NK cells were found to be negative for CD69 , suggesting that they are in a dysfunctional state.

Taken together, several factors contribute to the development of an immunosuppressive microenvironment that may mediate major resistance to ICIs in GIST .

GIST has conducted multiple clinical and preclinical trials to examine the effects of immunotherapies such as cytokines, ICIs , antibodies, ADCs , vaccines and ACTs . Some of them are promising, while others are unsatisfactory, especially for ICIs .

The relatively low expression of PD1/PD-L1 and the absence of MHC-I in GIST may partly explain the hyporesponsiveness to ICIs , which also makes us look forward to the emergence of other new immunotherapeutic targets, such as M2 macrophages, Treg Cells, LAG-3 , Tim-3 , CTAs , WT-1 , and CSPG4 , deserve more attention.

Advances in immunotherapy for gastrointestinal stromal tumors

references:

1. Advances in immunology and immunotherapy for mesenchymal gastrointestinal cancers. Mol Cancer. 2023 Apr 18;22(1):71.

(source:internet, reference only)


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