Cell-cell proximity influences tissue homeostasis and disease progression, yet robust quantification across varying cell abundances remains challenging. We introduce a Monte Carlo simulation framework using the G-function as a spatial randomness reference to detect proximity differences between case groups independent of cell count. Three metrics, G-area, G-difference, and G-ratio, were evaluated for summarizing G-function outputs, alongside established approaches such as the Morisita-Horn Index and likelihood ratio. G-area most accurately captured group-level proximity changes. To demonstrate generalizability, we validated G-area in two external multiplex imaging datasets from colorectal and prostate cancer. This framework provides a cell-count-robust method for spatial analysis, enabling more reliable detection of microenvironmental changes across diseases and imaging platforms.

Abstract Multi-agent COJEC chemotherapy is the main-stay of induction treatment for patients diagnosed with high-risk neuroblastoma. However, at least 10% of patients will be primary refractory to chemotherapy and only 50% achieve 5-year overall survival. The bone marrow is the most frequent site of metastasis in these patients. Novel approaches are required to improve response rates but the inter- and intra-patient tumour heterogeneity and dynamics of the neuroblastoma immune microenvironment makes anticipation of resistance phenotypes incredibly challenging. We present here a novel immunocompetent C57 Bl/6 model of Th- ALK(F1174L)/MYCN neuroblastoma, in which spontaneous abdominal tumours are driven by expression of mutant Anaplastic Lymphoma Kinase and over-expression of Mycn in the neural crest. We have used this model to generate a personalised dosing schedule inducing COJEC-chemotherapy resistance, in which individual mice receive chemotherapy cycles dependent upon the progression of their neuroblastoma tumours. Using both scRNAseq and spatial immunophenotyping gave us extraordinary precision in our comprehensive analysis of the tumour intrinsic and microenvironmental factors associated with COJEC resistance. We found that the resistance phenotype was driven by Cdk8 upregulation in adrenergic and mesenchymal tumour cells. Infiltration of immunosuppressive myeloid-derived immune cells and remodeling of the tumour-associated stroma further contributed to COJEC resistance. In the bone marrow we observed expansion of neutrophils and evidence of NETosis associated with micro-metastatic disease. Our results further endorse the development of CDK8-targeting therapeutics for neuroblastoma patients which might boost the anti-tumour immune response. Additional studies will be required to define the roles of neutrophils and neutrophil NETosis in neuroblastoma progression and metastasis. Our C57 Bl/6 model will be pivotal in future preclinical studies of immune-modulating therapeutics.

BACKGROUND & AIMS:Targeted therapies for biliary tract cancers (BTC) are approved in the second-line setting. We investigated response to first-line platinum-based palliative systemic anti-cancer treatment in patients with the most common druggable alterations to determine the optimal positioning of targeted therapies in the treatment algorithm. METHODS:Patients treated at the Beatson West of Scotland centralized BTC clinic who underwent genomic profiling were retrospectively selected; those with the most common actionable alterations who received platinum-based treatment were included in clinical analyses. Observations were supported by tracking IDH1 mutations with digital droplet PCR on longitudinal cell-free DNA samples. RESULTS:Druggable alterations were identified in 39.7% of patients; HER2 overexpression/amplification and IDH1 R132 mutations were the most commonly identified alterations. Compared to patients with HER2-positive tumors (n = 11), patients with IDH1 R132 mutations (n = 6) had longer median time to best response (5.99 vs. 2.5 months; hazard ratio [HR] 0.28; 95% CI 0.09-0.91; p = 0.0033), and median time to progression (17.2 vs. 5.6 months; HR 0.266; 95% CI 0.0955-0.741; p = 0.0075). Compared to a cohort of patients without either alteration, those with IDH1-mutated BTC had longer median overall survival (HR 0.30; 95% CI 0.14-0.67; p = 0.0229); those with HER2-positive BTC showed a worse median time to progression (HR 1.97; 95% CI 0.85-4.58; p = 0.0408) and a trend towards worse median overall survival (HR 1.38; 95% CI 0.61-3.13; p = 0.3884). IDH1 variant allele frequency decreased during first-line treatment, regardless of response; prolonged benefit from ivosidenib in the second line was observed when variant allele frequency was <1 at the start of targeted treatment. CONCLUSIONS:These preliminary data suggest that targeted therapies may need to be introduced earlier, including in the first-line setting, with strategies tailored to specific molecular alterations. Access to platinum-based palliative systemic anti-cancer treatment remains particularly important for patients with IDH1-mutated BTC. IMPACT AND IMPLICATIONS:We conducted an analysis to determine whether patients with biliary tract cancer harboring the most common actionable alterations (i.e. IDH1 R132 mutations and HER2 overexpression or amplification) had different patterns of response to first-line platinum-based chemo(immuno)therapy. The goal was to determine whether the introduction of targeted therapies in the first-line setting should be tailored based on the alteration detected. In our cohort, patients with IDH1 R132 mutations experienced durable benefit from platinum-based chemotherapy, while those with HER2-positive tumors only experienced a short-lived benefit from it. Although these findings are exploratory and limited by small sample size, they may inform future strategies for earlier integration of targeted therapies and support the design of prospective clinical trials.

Abstract Purpose: We tested whether circulating tumor DNA (ctDNA) changes may be used to assess early response and clinical outcomes in patients with metastatic colorectal cancer (mCRC) undergoing first-line systemic anticancer therapy (SACT). Experimental Design: Eight hundred sixty-two plasma samples were collected 4-weekly from baseline (BL) until disease progression in patients with mCRC receiving first-line SACT. ctDNA was tested using tissue-agnostic next-generation sequencing panels. ctDNA normalization was defined as ≥99% clearance after 1 month of therapy (Mo1) in the three variants with the highest allele frequency in BL ctDNA. Results: Eighty-three paired samples from 75 patients were available for analysis. Twelve pairs (14.4%) showed no variants in either BL or Mo1. In the remaining 71 comparisons (65 patients), 37 (52.1%) showed ctDNA normalization at Mo1. Patients who cleared ctDNA had significantly longer overall (45.6 months) and progression-free survival (13.9 months) compared with nonnormalized patients [overall survival = 22.6 months (log-rank P = 0.01) and progression-free survival = 10.7 months (log-rank P = 0.036), respectively]. In addition, a higher response rate was observed in patients with ctDNA clearance (72.9%) compared with nonnormalized cases (38.2%). Longitudinal sequencing of at least four time points in patients with a progression-free survival of >10 months showed emerging variants in 47.8% of cases; in all these patients, the trajectory of these new “outlier” variants seemed in stark contrast with the clinical–radiological course of disease and the trend in other mutations. Conclusions: ctDNA clearance represents an early indicator of benefit from SACT in patients with mCRC; serial tracking of multiple variants is warranted to improve specificity and avoid misleading information due to the emergence of mutations of unknown clinical significance.

Supplementary Table S1. Clinical characteristics of the cohorts used in the circulating biomarker analysis Supplementary Table S2. Cox proportional hazard regression analysis for miRNAs whose deregulation is associated with PFS Supplementary Table S3. Cox proportional hazard regression analysis for miRNAs whose deregulation is associated with OS Supplementary Table S4A. Multivariate Analysis of Factors Affecting Progression Free Survival in the PROSPECT-R Trial Supplementary Table S4B. Multivariate Analysis of Factors Affecting Overall Survival in the PROSPECT-R Trial Supplementary Table S5. Associaiton between MIR652-3p expression and overall response rate (ORR) in the PROSPECT-R Trial Supplementary Table S6. Toxicity, dose reductions and treatment discontinuation Supplementary Table S7. Backward stepwise selection of covariate for the pooled analysis. Supplementary Table S8. MVA prognostic model based on known prognostic features in patients treated with regorafenib. Supplementary Table S9. Comparison between MIR652-3p expression by ISH and by RT-PCR in tissues from patients enrolled in the PROSPECT-R Trial Supplementary Table S10. Response after 8 weeks of treatment according to MIR652-3p ISH score in the PROSPECT-R Trial Supplementary Table S11. Comparison between MIR652-3p expression in tissue versus liquid biopses in tissues from patients enrolled in the PROSPECT-R Trial Supplementary Table S12. Clinical-pathological features of the PDO biobank Table S13. List of deregulated genes in the comparison between treated and untreated EC cells grown in the 1021BL co-culture Table S14. List of deregulated genes in the comparison between EC cells grown in the 1021BL Versus the 1021PD co-culture Table S15. List of deregulated genes in the comparison between treated and untreated CAFs grown in the 1021BL co-culture Table S16. List of deregulated genes in the comparison between treated and untreated PDOs grown in the 1021BL co-culture Table S17. List of deregulated genes in the comparison between PDOs grown in the 1021BL Versus the 1021PD co-culture Table S18. List of deregulated genes in the comparison between CAFs grown in the 1021BL Versus the 1021PD co-culture Table S19. List of deregulated genes in the comparioson between 1021BL MIR623-3p over-expressing PDOs and relative contorls

AbstractPurpose: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. Experimental Design: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. Results: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652–3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a “control” group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652–3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. Conclusions: Our results identify MIR652–3p as a potential biomarker and as a driver of cell and non–cell-autonomous mechanisms of resistance to regorafenib.

Cancer evolution lays the groundwork for predictive oncology. Testing evolutionary metrics requires quantitative measurements in controlled clinical trials. We mapped genomic intratumor heterogeneity in locally advanced prostate cancer using 642 samples from 114 individuals enrolled in clinical trials with a 12-year median follow-up. We concomitantly assessed morphological heterogeneity using deep learning in 1,923 histological sections from 250 individuals. Genetic and morphological (Gleason) diversity were independent predictors of recurrence (hazard ratio (HR) = 3.12 and 95% confidence interval (95% CI) = 1.34–7.3; HR = 2.24 and 95% CI = 1.28–3.92). Combined, they identified a group with half the median time to recurrence. Spatial segregation of clones was also an independent marker of recurrence (HR = 2.3 and 95% CI = 1.11–4.8). We identified copy number changes associated with Gleason grade and found that chromosome 6p loss correlated with reduced immune infiltration. Matched profiling of relapse, decades after diagnosis, confirmed that genomic instability is a driving force in prostate cancer progression. This study shows that combining genomics with artificial intelligence-aided histopathology leads to the identification of clinical biomarkers of evolution.

Supplementary Figure S6. MIR652-3p targets PSAT1 and ANGPT1

BackgroundThis study aimed to identify microRNAs (miRs) as circulating biomarkers of resistance to first-line trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.MethodsA high-throughput 1015 Exiqon miRCURY LNA™ microRNA inhibitor library screen was performed in trastuzumab-treated HER2-positive NCI-N87 and HER2-negative FLO-1 oesophago-gastric cancer cell lines. NanoString nCounter® miR analysis was performed in NCI-N87, FLO-1, and MAGIC trial (ISRCTN93793971) formalin-fixed paraffin-embedded (FFPE) oesophago-gastric cancer patient samples. MiR-148a-3p copies in plasma samples were quantified using digital droplet polymerase chain reaction (ddPCR) from HER2-positive oesophago-gastric cancer patients treated with standard-of-care trastuzumab-based therapy within the FOrMAT (NCT02112357) and PLATFORM (NCT02678182) clinical trials. The primary endpoints were overall survival (OS) for plasma miR-148a-3p HIGH (>median) versus LOW (≤median). The secondary endpoints were progression-free survival (PFS) and 3-month progression-free rates (PFRs) miR-148a-3p HIGH versus LOW. PLATFORM sensitivity analysis normalised miR-148a-3p (NmiR-148a-3p).ResultsThe inhibition of miR-148a-3p reduced NCI-N87 relative cell viability (<0.6) and expression was high (>242) in NCI-N87 and HER2-positive MAGIC trial patients (n=5). Normalised-miR-148a-3p (NmiR-148a-3p) LOW versus HIGH demonstrated a statistically significant difference in 3-month PFRs (n=23; OR, 0.11 [0.02–0.78]; p=0.027; aOR, 0.03 [0.001–0.71], p=0.029) but no difference in OS or PFS. There was no statistically significant relationship between miR-148-3p LOW versus HIGH for OS (PLATFORM, n=62; hazard ratio [HR], 0.98 [0.57–1.66]; p=0.933; FOrMAT, n=8; HR, 0.54 [0.13–2.31]; p=0.322), PFS (n=62; HR, 1.08 [0.65–1.81]; p=0.759; FOrMAT, n=8; HR, 1.26 [0.31–5.07]; p=0.714), or PFRs (PLATFORM, n=31; odds ratio [OR], 0.67 [0.2–2.8]; p=0.577).ConclusionNormalised miR-148a-3p may be a relevant biomarker for trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.

Supplementary methods and Figures S1-S17

Junctional adhesion molecules (JAMs) play a critical role in cell permeability, polarity and migration. JAM-A, a key protein of the JAM family, is altered in a number of conditions including cancer; however, consequences of JAM-A dysregulation on carcinogenesis appear to be tissue dependent and organ dependent with significant implications for the use of JAM-A as a biomarker or therapeutic target. Here, we test the expression and prognostic role of JAM-A downregulation in primary and metastatic colorectal cancer (CRC) ( n = 947). We show that JAM-A downregulation is observed in ~60% of CRC and correlates with poor outcome in four cohorts of stages II and III CRC ( n = 1098). Using JAM-A knockdown, re-expression and rescue experiments in cell line monolayers, 3D spheroids, patient-derived organoids and xenotransplants, we demonstrate that JAM-A silencing promotes proliferation and migration in 2D and 3D cell models and increases tumour volume and metastases in vivo. Using gene-expression and proteomic analyses, we show that JAM-A downregulation results in the activation of ERK, AKT and ROCK pathways and leads to decreased bone morphogenetic protein 7 expression. We identify MIR21 upregulation as the cause of JAM-A downregulation and show that JAM-A rescue mitigates the effects of MIR21 overexpression on cancer phenotype. Our results identify a novel molecular loop involving MIR21 dysregulation, JAM-A silencing and activation of multiple oncogenic pathways in promoting invasiveness and metastasis in CRC.

Introduction: Monoclonal antibodies against the Epidermal Growth Factor Receptor (EGFR) such as cetuximab or panitumumab are used for the treatment of metastatic colorectal cancer (mCRC) patients. Unfortunately, most patients develop resistance against these therapies within months. Several studies have shown that aberrations in the RAS pathway are responsible for resistance. However, even in metastases that are refractory to anti-EGFR treatment a significant fraction of RAS wild-type (wt) cells remain. These findings suggest a cross-talk between RAS mutant and wt cells in mediating resistance in the wt compartment. Methods: Mouse and patient-derived organoids from mCRC as well as CRC cell lines were used to test the contribution of extracellular vesicles in mediating resistance in RAS wt cells. Using conditioned media, transfection experiments and liquid biopsies (plasma and urine) from patients differential expression of the let-7g microRNA was demonstrated in microvesicles from cetuximab sensitive and resistant cells. Changes in expression of let-7g were further analyzed by in-situ hybridization in tissues. Results: Conditioned media from RAS mutant organoids rendered RAS wt organoids resistant against cetuximab treatment. Basal let-7g expression from pre-treatment plasma and urine samples of RAS wt patients correlated with clinical outcome and changes in let-7g circulating levels mirrored clinical behavior. In-situ hybridization in tissues confirmed changes in expression of the let-7g microRNA observed in plasma and urine samples. Conclusions: Our data suggest that let-7g microRNA might function as a paracrine mediator of anti-EGFR resistance and might be exploited as a non-invasive biomarker of resistance to cetuximab treatment. Further work is ongoing to characterize the molecular mechanisms underpinning let-7g mediated effect on anti-EGFR sensitivity in RAS wt CRC cells. Citation Format: Jens C. Hahne, Andrea Lampis, Michele Ghidini, Margherita Ratti, Chiara Senti, Rodolfo Passalacqua, Luciano Cascione, Chiara Braconi, Owen Sansom, Matteo Fassan, Nicola Valeri. Expression of exosomal let-7g in biofluids and outcome in colon cancer patient treated with anti-EGFR therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2373.

Introduction Despite the efforts of the scientific community, the prognosis of metastatic colorectal cancer (mCRC) remains poor. Actionable gene fusions such as Neurotrophic Tropomyosin Receptor Kinases (NTRK) rearrangements are rare but might represent a new target to improve outcomes in this setting. The first-generation TRK inhibitors, larotrectinib and entrectinib, have demonstrated efficacy and safety in mCRC cancer patients exhibiting NTRK pathogenic fusions. Moreover, second-generation molecules are emerging, able to overcome the acquired resistance to NTRK blocking. Areas covered This review aims to report the current knowledge and the available evidence on NTRK fusion in mCRC, with a focus on molecular bases, clinical characteristics, prognostic meaning, and new therapeutic approaches, from the perspective of the clinical oncologist. Expert opinion Considering the limited options associated with the treatment of mCRC patients, the possibility of identifying new molecular biomarkers is an urgent clinical need. The availability of new molecular targets and the combinations of different agents might represent the true breakthrough point, allowing for change in the clinical course of colorectal cancer patients.

Oesophageal cancer is one of the most aggressive malignancies with limited treatment options, thus resulting in a high morbidity and mortality. With 5-year survival rates of only 5-10%, oesophageal cancer holds a dismal prognosis for patients. In order to improve overall survival, the early diagnosis and tools for patient stratification for personalized treatment are urgent needs. A minority of oesophageal cancers belong to the spectrum of Lynch syndrome-associated cancers and are characterized by microsatellite instability (MSI). Microsatellite instability is a consequence of defective mismatch repair protein functions and it has been well characterized in other gastrointestinal tumours, such as colorectal and gastric cancer. In the latter, high levels of MSI are associated with a better prognosis and with an increased benefit to immune-based therapies. Therefore, similar therapeutic approaches could offer an opportunity of treatment for oesophageal cancer patients with MSI. Apart from immune checkpoint inhibitors, other immunotherapies such as adoptive T-cell transfer, peptide vaccine and oncolytic viruses are under investigation in oesophageal cancer patients. In the present review, the rationale and current knowledge about immunotherapies in oesophageal cancer are summarised.