Cancer response to immunotherapy depends on the infiltration of CD8⁺ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8⁺ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8⁺ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival.

The molecular characterisation of medulloblastoma, the most common paediatric brain tumour, is crucial for the correct management and treatment of this heterogenous disease. However, insufficient tissue sample, the presence of tumour heterogeneity, or disseminated disease can challenge its diagnosis and monitoring. Here, we report that the cerebrospinal fluid (CSF) circulating tumour DNA (ctDNA) recapitulates the genomic alterations of the tumour and facilitates subgrouping and risk stratification, providing valuable information about diagnosis and prognosis. CSF ctDNA also characterises the intra-tumour genomic heterogeneity identifying small subclones. ctDNA is abundant in the CSF but barely present in plasma and longitudinal analysis of CSF ctDNA allows the study of minimal residual disease, genomic evolution and the characterisation of tumours at recurrence. Ultimately, CSF ctDNA analysis could facilitate the clinical management of medulloblastoma patients and help the design of tailored therapeutic strategies, increasing treatment efficacy while reducing excessive treatment to prevent long-term secondary effects.

The levels of cell free circulating tumor DNA (ctDNA) in plasma correlated with treatment response and outcome in systemic lymphomas. Notably, in brain tumors, the levels of ctDNA in the cerebrospinal fluid (CSF) are higher than in plasma. Nevertheless, their role in central nervous system (CNS) lymphomas remains elusive. We evaluated the CSF and plasma from 19 patients: 6 restricted CNS lymphomas, 1 systemic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic mutations of the primary tumor, then variant-specific droplet digital PCR was designed for each mutation. At time of enrolment, we found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. Conversely, plasma ctDNA was detected in only 2/6 patients with restricted CNS lymphoma with lower variant allele frequencies than CSF ctDNA. Moreover, we detected CSF ctDNA in 1 patient with CNS lymphoma in complete remission and in 1 patient with systemic lymphoma, 3 and 8 months before CNS relapse was confirmed; indicating CSF ctDNA might detect CNS relapse earlier than conventional methods. Finally, in 2 cases with CNS lymphoma, CSF ctDNA was still detected after treatment even though a complete decrease in CSF tumor cells was observed by flow cytometry (FC), indicating CSF ctDNA better detected residual disease than FC. In conclusion, CSF ctDNA can better detect CNS lesions than plasma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS and systemic lymphomas.

Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8⁺ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.

The correct characterisation of central nervous system (CNS) malignancies is crucial for accurate diagnosis and prognosis and also the identification of actionable genomic alterations that can guide the therapeutic strategy. Surgical biopsies are performed to characterise the tumour; however, these procedures are invasive and are not always feasible for all patients. Moreover, they only provide a static snapshot and can miss tumour heterogeneity. Currently, monitoring of CNS cancer is performed by conventional imaging techniques and, in some cases, cytology analysis of the cerebrospinal fluid (CSF); however, these techniques have limited sensitivity. To overcome these limitations, a liquid biopsy of the CSF can be used to obtain information about the tumour in a less invasive manner. The CSF is a source of cell-free circulating tumour DNA (ctDNA), and the analysis of this biomarker can characterise and monitor brain cancer. Recent studies have shown that ctDNA is more abundant in the CSF than plasma for CNS malignancies and that it can be sequenced to reveal tumour heterogeneity and provide diagnostic and prognostic information. Furthermore, analysis of longitudinal samples can aid patient monitoring by detecting residual disease or even tracking tumour evolution at relapse and, therefore, tailoring the therapeutic strategy. In this review, we provide an overview of the potential clinical applications of the analysis of CSF ctDNA and the challenges that need to be overcome in order to translate research findings into a tool for clinical practice.

TIN2 is central to the shelterin complex, linking the telomeric proteins TRF1 and TRF2 with TPP1/POT1. Mutations in TINF2, which encodes TIN2, that are found in dyskeratosis congenita (DC) result in very short telomeres and cluster in a region shared by the two TIN2 isoforms, TIN2S (short) and TIN2L (long). Here we show that TIN2L, but not TIN2S, is phosphorylated. TRF2 interacts more with TIN2L than TIN2S, and both the DC cluster and phosphorylation promote this enhanced interaction. The binding of TIN2L, but not TIN2S, is affected by TRF2-F120, which is also required for TRF29s interaction with end processing factors such as Apollo. Conversely, TRF1 interacts more with TIN2S than with TIN2L. A DC-associated mutation further reduces TIN2L-TRF1, but not TIN2S-TRF1, interaction. Cells overexpressing TIN2L or phosphomimetic TIN2L are permissive to telomere elongation, whereas cells overexpressing TIN2S or phosphodead TIN2L are not. Telomere lengths are unchanged in cell lines in which TIN2L expression has been eliminated by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated mutation. These results indicate that TIN2 isoforms are biochemically and functionally distinguishable and that shelterin composition could be fundamentally altered in patients with TINF2 mutations.