Osimertinib is associated with improved outcomes in pre-treated non-small cell lung cancer leptomeningeal metastases: A systematic review and meta-analysiss.

Purpose: Leptomeningeal metastasis (LM) is a severe complication of non-small cell lung cancer (NSCLC). In patients with NSCLC LM harboring epidermal growth factor receptor (EGFR) mutations, osimertinib is favored over alternative EGFR tyrosine kinase inhibitors (TKIs). However, the efficacy of osimertinib relative to other EGFR-TKIs is not well established for patients with LM. We aimed to compare the efficacy of EGFR-TKIs in EGFR-mutated NSCLC LM. Methods: This systematic review and meta-analysis performed according to PRISMA guidelines included studies of adult patients with EGFR-mutated NSCLC and a diagnosis of LM who received an EGFR-TKI for the treatment of LM. We searched Medline ALL, Embase, Cochrane Central Register of Controlled Trials, Scopus, and Web of Science Core Collection. The evaluation of biases was done by using the Ottawa-Newscastle scale. The hazard ratio was used as the parameter of interest for overall survival (OS) and central nervous system-specific progression-free survival (PFS). Results: 128 publications were included with 243 patients and 282 lines of EGFR-TKI for NSCLC LM that met inclusion criteria. The median PFS in patients receiving any EGFR-TKI was 9.1 months, and the median OS was 14.5 months. In univariate analyses of the entire cohort, osimertinib treatment demonstrated significantly prolonged PFS, but not OS, compared to other EGFR-TKIs. Osimertinib demonstrated significantly prolonged PFS and OS in the subset of patients who were previously treated with EGFR-TKIs, but not in EGFR-TKI naïve patients. Conclusion: Osimertinib is associated with improved outcomes compared to other EGFR-TKIs, particularly in patients previously treated with EGFR-TKIs. An important limitation is that most patients were derived from retrospective reports. These results highlight the need for prospective studies for this difficult-to-treat patient population.

Invasive growth of brain metastases is linked to CHI3L1 release from pSTAT3-positive astrocytes.

BACKGROUND: Compared to minimally invasive brain metastases (MI BrM), highly invasive (HI) lesions form abundant contacts with cells in the peritumoral brain parenchyma and are associated with poor prognosis. Reactive astrocytes (RAs) labeled by phosphorylated STAT3 (pSTAT3) have recently emerged as a promising therapeutic target for BrM. Here, we explore whether BrM invasion pattern is influenced by pSTAT3+ RAs and may serve as a predictive biomarker for STAT3 inhibition. METHODS: We used immunohistochemistry to identify pSTAT3+ RAs in HI and MI human and patient-derived xenograft (PDX) BrM. Using PDX, syngeneic, and transgenic mouse models of HI and MI BrM, we assessed how pharmacological STAT3 inhibition or RA-specific STAT3 genetic ablation affected BrM growth in vivo. Cancer cell invasion was modeled in vitro using a brain slice-tumor co-culture assay. We performed single-cell RNA sequencing of human BrM and adjacent brain tissue. RESULTS: RAs expressing pSTAT3 are situated at the brain-tumor interface and drive BrM invasive growth. HI BrM invasion pattern was associated with delayed growth in the context of STAT3 inhibition or genetic ablation. We demonstrate that pSTAT3+ RAs secrete Chitinase 3-like-1 (CHI3L1), which is a known STAT3 transcriptional target. Furthermore, single-cell RNA sequencing identified CHI3L1-expressing RAs in human HI BrM. STAT3 activation, or recombinant CHI3L1 alone, induced cancer cell invasion into the brain parenchyma using a brain slice-tumor plug co-culture assay. CONCLUSIONS: Together, these data reveal that pSTAT3+ RA-derived CHI3L1 is associated with BrM invasion, implicating STAT3 and CHI3L1 as clinically relevant therapeutic targets for the treatment of HI BrM.

Intrathecal trastuzumab versus alternate routes of delivery for HER2-targeted therapies in patients with HER2+ breast cancer leptomeningeal metastases.

BACKGROUND: Patients with HER2+ breast cancer (BC) frequently develop leptomeningeal metastases (LM). While HER2-targeted therapies have demonstrated efficacy in the neoadjuvant, adjuvant, and metastatic settings, including for parenchymal brain metastases, their efficacy for patients with LM has not been studied in a randomized controlled trial. However, several single-armed prospective studies, case series and case reports have studied oral, intravenous, or intrathecally administered HER2-targeted therapy regimens for patients with HER2+ BC LM. METHODS: We conducted a systematic review and meta-analysis of individual patient data to evaluate the efficacy of HER2-targeted therapies in HER2+ BC LM in accordance with PRISMA guidelines. Targeted therapies evaluated were trastuzumab (intrathecal or intravenous), pertuzumab, lapatinib, neratinib, tucatinib, trastuzumab-emtansine and trastuzumab-deruxtecan. The primary endpoint was overall survival (OS), with CNS-specific progression-free survival (PFS) as a secondary endpoint. RESULTS: 7780 abstracts were screened, identifying 45 publications with 208 patients, corresponding to 275 lines of HER2-targeted therapy for BC LM which met inclusion criteria. In univariable and multivariable analyses, we observed no significant difference in OS and CNS-specific PFS between intrathecal trastuzumab compared to oral or intravenous administration of HER2-targeted therapy. Anti-HER2 monoclonal antibody-based regimens did not demonstrate superiority over HER2 tyrosine kinase inhibitors. In a cohort of 15 patients, treatment with trastuzumab-deruxtecan was associated with prolonged OS compared to other HER2-targeted therapies and compared to trastuzumab-emtansine. CONCLUSIONS: The results of this meta-analysis, comprising the limited data available, suggest that intrathecal administration of HER2-targeted therapy for patients with HER2+ BC LM confers no additional benefit over oral and/or IV treatment regimens. Although the number of patients receiving trastuzumab deruxtecan in this cohort is small, this novel agent offers promise for this patient population and requires further investigation in prospective studies.

Single-cell spatial landscapes of the lung tumour immune microenvironment.

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution1-9. Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice.

Single-cell spatial immune landscapes of primary and metastatic brain tumours.

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell-cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

Invasive growth associated with cold-inducible RNA-binding protein expression drives recurrence of surgically resected brain metastases.

BACKGROUND: Sixty percent of surgically resected brain metastases (BrM) recur within 1 year. These recurrences have long been thought to result from the dispersion of cancer cells during surgery. We tested the alternative hypothesis that invasion of cancer cells into the adjacent brain plays a significant role in local recurrence and shortened overall survival. METHODS: We determined the invasion pattern of 164 surgically resected BrM and correlated with local recurrence and overall survival. We performed single-cell RNA sequencing (scRNAseq) of >15,000 cells from BrM and adjacent brain tissue. Validation of targets was performed with a novel cohort of BrM patient-derived xenografts (PDX) and patient tissues. RESULTS: We demonstrate that invasion of metastatic cancer cells into the adjacent brain is associated with local recurrence and shortened overall survival. scRNAseq of paired tumor and adjacent brain samples confirmed the existence of invasive cancer cells in the tumor-adjacent brain. Analysis of these cells identified cold-inducible RNA-binding protein (CIRBP) overexpression in invasive cancer cells compared to cancer cells located within the metastases. Applying PDX models that recapitulate the invasion pattern observed in patients, we show that CIRBP is overexpressed in highly invasive BrM and is required for efficient invasive growth in the brain. CONCLUSIONS: These data demonstrate peritumoral invasion as a driver of treatment failure in BrM that is functionally mediated by CIRBP. These findings improve our understanding of the biology underlying postoperative treatment failure and lay the groundwork for rational clinical trial development based upon invasion pattern in surgically resected BrM.

RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.

A gain-of-functional screen identifies the Hippo pathway as a central mediator of receptor tyrosine kinases during tumorigenesis.

The Hippo pathway has emerged as a key signaling pathway that regulates various biological functions. Dysregulation of the Hippo pathway has been implicated in a broad range of human cancer types. While a number of stimuli affecting the Hippo pathway have been reported, its upstream kinase and extracellular regulators remain largely unknown. Here we performed the first comprehensive gain-of-functional screen for receptor tyrosine kinases (RTKs) regulating the Hippo pathway using an RTK overexpression library and a Hippo signaling activity biosensor. Surprisingly, we found that the majority of RTKs could regulate the Hippo signaling activity. We further characterized several of these novel relationships [TAM family members (TYRO3, AXL, METRK), RET, and FGFR family members (FGFR1 and FGFR2)] and found that the Hippo effectors YAP/TAZ are central mediators of the tumorigenic phenotypes (e.g., increased cell proliferation, transformation, increased cell motility, and angiogenesis) induced by these RTKs and their extracellular ligands (Gas6, GDNF, and FGF) through either PI3K or MAPK signaling pathway. Significantly, we identify FGFR, RET, and MERTK as the first RTKs that can directly interact with and phosphorylate YAP/TAZ at multiple tyrosine residues independent of upstream Hippo signaling, thereby activating their functions in tumorigenesis. In conclusion, we have identified several novel kinases and extracellular stimuli regulating the Hippo pathway. Our findings also highlight the pivotal role of the Hippo pathway in mediating Gas6/GDNF/FGF-TAM/RET/FGFR-MAPK/PI3K signaling during tumorigenesis and provide a compelling rationale for targeting YAP/TAZ in RTK-driven cancers.

GGA3-mediated recycling of the RET receptor tyrosine kinase contributes to cell migration and invasion.

The RET receptor tyrosine kinase plays important roles in regulating cellular proliferation, migration, and survival in the normal development of neural crest derived tissues. However, aberrant activation of RET, through oncogenic mutations or overexpression, can contribute to tumourigenesis, regional invasion, and metastasis of several human cancers. RET is expressed as two main isoforms with unique C-terminal sequences that differ in protein interactions and subcellular trafficking in response to RET activation, and which also have distinct oncogenic potentials. The long isoform, termed RET51, is internalized from the membrane in response to stimulation by its ligand, GDNF, but is known to recycle back to the surface via RAB11 endosomes. However, the mechanisms regulating this process and its cellular effects have not been defined. Here, we show that recycling of RET51 requires a multicomponent complex that includes the endosomal-sorting protein GGA3, which mediates GDNF-dependent slow recycling of RET51 receptors to the plasma membrane. Our data show that the GRB2 adapter associates with RET51 through interactions with its C-terminal sequences, facilitating recruitment of active ARF6 and GGA3 interaction, and that depletion of GGA3 or ARF6 reduced RET51 recycling. Further, GGA3 knockdown accelerated RET51 degradation and also attenuated RET-mediated AKT activation. Finally, we showed that recycling of RET51 to the cell surface through association with GGA3 and ARF6 contributes to RET51-dependent cell motility, migration, and invasion. Our data establish RET recycling as a mechanism coordinating location and duration of RET signals in order to direct cell movement and invasion.

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Differential roles of RET isoforms in medullary and papillary thyroid carcinomas.

The RET receptor tyrosine kinase mediates cell proliferation, survival and migration in embryogenesis and is implicated in the transformation and tumour progression in multiple cancers. RET is frequently mutated and constitutively activated in familial and sporadic thyroid carcinomas. As a result of alternative splicing, RET is expressed as two protein isoforms, RET9 and RET51, which differ in their unique C-terminal amino acids. These isoforms have distinct intracellular trafficking and associated signalling complexes, but functional differences are not well defined. We used shRNA-mediated knockdown (KD) of individual RET isoforms or of total RET to evaluate their functional contributions in thyroid carcinoma cells. We showed that RET is required for cell survival in medullary (MTC) but not papillary thyroid carcinoma (PTC) cells. In PTC cells, RET depletion reduced cell migration and induced a flattened epithelial-like morphology. RET KD decreased the expression of mesenchymal markers and matrix metalloproteinases and reduced anoikis resistance and invasive potential. Further, we showed that RET51 depletion had significantly greater effects on each of these processes than RET9 depletion in both MTC and PTC cells. Finally, we showed that expression of RET, particularly RET51, was correlated with malignancy in a panel of human thyroid tumour tissues. Together, our data show that RET expression promotes a more mesenchymal phenotype with reduced cell-cell adhesion and increased invasiveness in PTC cell models, but is more important for tumour cell survival, proliferation and anoikis resistance in MTC models. Our data suggest that the RET51 isoform plays a more prominent role in mediating these processes compared to RET9.