Biopsies Of Cancer Patients Research Articles

Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

Collagen plays a critical role in regulating breast cancer progression and therapeutic resistance. An improved understanding of both the features and drivers of tumor-permissive and -restrictive collagen matrices are critical to improve prognostication and develop more effective therapeutic strategies. In this study, using a combination of in vitro, in vivo and bioinformatic experiments, we show that type III collagen (Col3) plays a tumor-restrictive role in human breast cancer. We demonstrate that Col3-deficient, human fibroblasts produce tumor-permissive collagen matrices that drive cell proliferation and suppress apoptosis in non-invasive and invasive breast cancer cell lines. In human triple-negative breast cancer biopsy samples, we demonstrate elevated deposition of Col3 relative to type I collagen (Col1) in non-invasive compared to invasive regions. Similarly, bioinformatics analysis of over 1000 breast cancer patient biopsies from The Cancer Genome Atlas BRCA cohort revealed that patients with higher Col3:Col1 bulk tumor expression had improved overall, disease-free, and progression-free survival relative to those with higher Col1:Col3 expression. Using an established 3D culture model, we show that Col3 increases spheroid formation and induces the formation of lumen-like structures that resemble non-neoplastic mammary acini. Finally, our in vivo study shows co-injection of murine breast cancer cells (4T1) with rhCol3-supplemented hydrogels limits tumor growth and decreases pulmonary metastatic burden compared to controls. Taken together, these data collectively support a tumor-suppressive role for Col3 in human breast cancer and suggest that strategies that increase Col3 may provide a safe and effective therapeutic modality to limit recurrence in breast cancer patients.

Lack of TGFβ signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade.

Transforming growth factor beta (TGFβ) is well-recognized as an immunosuppressive player in the tumor microenvironment but also has a significant impact on cancer cell phenotypes. Loss of TGFβ signaling impairs DNA repair competency, which is described by a transcriptomic score, βAlt. Cancers with high βAlt have more genomic damage and are more responsive to genotoxic therapy. The growing appreciation that cancer DNA repair deficits are important determinants of immune response prompted us to investigate the association of βAlt with response to immune checkpoint blockade (ICB). We predicted that high βAlt tumors would be infiltrated with lymphocytes because of DNA damage burden and hence responsive to ICB. We analyzed public transcriptomic data from clinical trials and preclinical models using transcriptomic signatures of TGFβ targets, DNA repair genes, tumor educated immune cells and interferon. A high βAlt, immune poor mammary tumor derived transplant model resistant to programmed death ligand 1 (PD-L1) antibodies was studied using multispectral flow cytometry to interrogate the immune system. Metastatic bladder patients in IMvigor 210 who responded to ICB had significantly increased βAlt scores and experienced significantly longer overall survival compared to those with low βAlt scores (hazard ratio 0.62, P=0.011) . Unexpectedly, 75% of high βAlt cancers were immune poor as defined by low expression of tumor educated immune cell and interferon signatures. The association of high βAlt with immune poor cancer was also evident in TCGA and preclinical cancer models. We used a high βAlt, immune poor cancer to test therapeutic strategies to overcome its inherent anti-PD-L1 resistance. Combination treatment with radiation and TGFβ inhibition were necessary for lymphocytic infiltration and activated NK cells were required for ICB response. Bioinformatic analysis identified high βAlt, immune poor B16 and CT26 preclinical models and paired biopsies of cancer patients that also demonstrated NK cell activation upon response to ICB. Our studies support βAlt as a biomarker that predicts response to ICB albeit in immune poor cancers, which has implications for the development of therapeutic strategies to increase the number of cancer patients who will benefit from immunotherapy.

Abstract 2739: XON27, a novel and potent immunotherapy against hematologic malignancies

Abstract Background: Over the past decades, intensive research has led to the development of innovative therapies (monoclonal and bispecific antibodies, CAR T cells) that have increased the survival rate of patients with multiple myeloma (MM) and B cell lymphoma. However, despite this progress, the majority of patients relapse and become refractory due to the development of resistance. Polyclonal antibodies, with their ability to target multiple antigens, can counteract this resistance. Here we evaluate the safety and efficacy of XON27, a first-in-class glyco-humanized polyclonal antibody (GH-pAb), in MM and other blood cancers. Material and Methods: The ability of XON27 to induce antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and apoptosis was tested in a panel of hematological malignancy cells and peripheral blood mononuclear cells (PBMC). Specific binding to human blood cancers was assessed using tissue microarrays. To study resistance mechanisms, MM cell lines were cocultured with macrophages in the presence or absence of XON27 and macrophage. To study relapse mechanisms and tumorigenicity, repeated in vitro administration of XON27 was evaluated in KMS-12-BM. The in vivo efficacy of XON27 was evaluated in KMS-12-BM NMRI nude mice model. Pharmacokinetics and safety of this drug were assessed in non-human primate after repeated IV dosing up to 60mg/kg.Results: XON27 induces potent and specific cytotoxicity in several blood malignancy cell lines including human MM cells via multiple mechanisms: CDC (EC50=35.84 ug/ml), apoptosis (IC50=100 ug/ml) and ADCP (up to 40% of cell death at 50 ug/ml). The cytotoxicity induced by XON27 in the KMS-12-BM was significantly more potent compared to daratumumab (anti-CD38) in both CDC and apoptosis assays, XON27 was able to kill up to 100% of cancer cells without affecting PBMC. XON27 showed cross-reactivity with blood cancer patient biopsies without staining healthy tissue. In addition, repeated administrations of XON27 demonstrate its ability to effectively kill tumor cells after each administration. No resistance is induced after repeated treatments. Interestingly, XON27 blocked the proliferation of residual tumor cells for up to 45 days after the second treatment at 50 ug/ml compared to the control condition, demonstrating sustainable effect of XON27 on tumorigenicity. The in vitro efficacy of XON27 was translated into in vivo efficacy in a murine MM xenograft model. It induced a significant reduction in tumor growth of up to 75%. XON27 was also characterized by a high tolerability profile and satisfactory exposure in primates. Conclusion: Although many therapeutic advances have been made in the field of blood cancers, myeloma remains an incurable disease with a real need for new innovative therapies. XON27 represents a promising and selective immunotherapy which may provide an effective and safe treatment against refractory myeloma. Citation Format: Carine Ciron, Pierre Morice, Françoise Shneiker, Odile Duvaux, Firas Bassissi. XON27, a novel and potent immunotherapy against hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2739.

Targeting eIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A with zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages toward an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon (IFN) response uniformly across models. The induction of an IFN response is partially due to the inhibition of Sox4 translation by zotatifin. A similar induction of IFN-stimulated genes was observed in breast cancer patient biopsies following zotatifin treatment. Surprisingly, zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened IFN response, resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for zotatifin, and provide a rationale for new combination regimens consisting of zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

Bilayer Microfluidic Device for Combinatorial Plug Production.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

TMIC-68. SIRPΑ BLOCKADE AS AN IMMUNOTHERAPEUTIC STRATEGY TO TREAT BREAST CANCER BRAIN METASTASIS

Abstract Triple-negative breast cancer (TNBC) has a high incidence of brain metastasis, with no standard therapeutic strategy for treatment. Therefore, new targets are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, direct targeting SIRPα as a potential approach to mitigate metastatic burden remains understudied. Single-cell-RNA-sequencing indicates that SIRPα expression on basal epithelial cells, and TCGA data shows SIRPα expression is associated with a significant reduction in survival in basal-like breast cancer. Our immune staining against SIRPα in breast cancer patient biopsies shows a 3.5-fold increase in expression in metastatic lesions compared to the primary tumor (n=19; p ≤ 0.01). Staining of 4T1 parental and brain-trophic 4T1-Br3 cells showed an 84% increase in SIRPα in the metastatic cells (n=3; p ≤ 0.05). Furthermore, Real-time cell impedance analysis revealed that SIRPα blockade inhibits TNBC 4T1br3 cell migration (n=4; p≤0.01), suggesting a link between SIRPα expression and metastatic potential. Furthermore, in vivo SIRPα blockade reduced brain metastatic burden by approximately 90%, (n=4-7; p ≤ 0.05). Digital spatial profiling of tissues revealed that SIRPα blockade was associated with reductions in immune checkpoints, including CTLA-4, PD-1, PD-L1, and LAG3, and immunosuppressive T-cell/monocyte populations. Furthermore, our spatial analysis indicated that brain lesions of WT mice have increases in the extracellular matrix protein fibronectin in the immune enriched regions, potentially serving as a barrier for immune cell infiltration contributing to invasion, metastasis, and immune evasion, was reduced by 70% in SIRPα treated brain lesions (n-3-6; p ≤ 0.05). Thus, these data suggest that SIRPα blockade may influence the tumor microenvironment to limit brain metastatic breast cancer growth and potentially enhance patient survival to metastatic brain disease.

Exploring Co-occurrence patterns and microbial diversity in the lung microbiome of patients with non-small cell lung cancer

BackgroundIt has been demonstrated in the literature that a dysbiotic microbiome could have a negative impact on the host immune system and promote disease onset or exacerbation. Co-occurrence networks have been widely adopted to identify biomarkers and keystone taxa in the pathogenesis of microbiome-related diseases. Despite the promising results that network-driven approaches have led to in various human diseases, there is a dearth of research pertaining to key taxa that contribute to the pathogenesis of lung cancer. Therefore, our primary goal in this study is to explore co-existing relationships among members of the lung microbial community and any potential gained or lost interactions in lung cancer.ResultsUsing integrative and network-based approaches, we integrated four studies assessing the microbiome of lung biopsies of cancer patients. Differential abundance analyses showed that several bacterial taxa are different between tumor and tumor-adjacent normal tissues (FDR adjusted p-value < 0.05). Four, fifteen, and twelve significantly different associations were found at phylum, family, and genus levels. Diversity analyses suggested reduced alpha diversity in the tumor microbiome. However, beta diversity analysis did not show any discernible pattern between groups. In addition, four distinct modules of bacterial families were detected by the DBSCAN clustering method. Finally, in the co-occurrence network context, Actinobacteria, Firmicutes, Bacteroidetes, and Chloroflexi at the phylum level and Bifidobacterium, Massilia, Sphingobacterium, and Ochrobactrum at the genus level showed the highest degree of rewiring.ConclusionsDespite the absence of statistically significant differences in the relative abundance of certain taxa between groups, it is imperative not to overlook them for further exploration. This is because they may hold pivotal central roles in the broader network of bacterial taxa (e.g., Bifidobacterium and Massilia). These findings emphasize the importance of a network analysis approach for studying the lung microbiome since it could facilitate identifying key microbial taxa in lung cancer pathogenesis. Relying exclusively on differentially abundant taxa may not be enough to fully grasp the complex interplay between lung cancer and the microbiome. Therefore, a network-based approach can offer deeper insights and a more comprehensive understanding of the underlying mechanisms.

#6294 UTILITY OF KIDNEY BIOPSY AND SPECTRUM OF BIOPSY PROVEN KIDNEY DISEASES IN PATIENTS WITH MALIGNANCY: A RETROSPECTIVE SINGLE-CENTER EXPERIENCE

Abstract Background and Aims Kidney biopsy may improve diagnostic accuracy and aid in the management of cancer patients with kidney diseases. However, literature on the utility of kidney biopsy in cancer patients and survivors is lacking. We aimed to evaluate the clinical characteristics of cancer patients who underwent kidney biopsy, as well as the histological spectrum of biopsy proven kidney diseases in this cohort. Method This was a single-center retrospective study of all patients who had native kidney biopsies at the Singapore General Hospital between 1st October 2015 to 31st December 2022. Demographic, clinical, laboratory and histological data were retrieved from electronic medical records to identify patients with solid-organ or hematological malignancies who underwent kidney biopsy. Results Out of 1208 patients who underwent kidney biopsy between 1st October 2015 to 31st December 2022, 116 (9.6%) patients had diagnoses of solid-organ or hematological malignancies. Approximately two-thirds (79/116; 68.1%) had solid-organ malignancies, of which 21.5% had metastatic disease at time of biopsy. Approximately one-third (36/116; 31.0%) had hematological malignancies, of which 10 (8.6%) had a hematological stem-cell transplant (HSCT). One (0.9%) patient had both solid-organ and hematological malignancies. Sixty-eight (58.6%) patients were in remission at time of biopsy, comprising of 52 (44.8%) and 16 (13.8%) patients with solid -organ and hematological malignancies, respectively. Median age at biopsy was 65.7 years (IQR 56.5, 71.4) and majority was female (61/116; 52.6%). Comorbidities of hypertension, type 2 diabetes mellitus, autoimmunity, and liver transplant were found in 36.2% (42/116), 23.3% (27/116), 8.6% (10/116) and 1.7% (2/116) patients respectively. Median serum creatinine at time of biopsy was 152.0μmol/L (IQR 101.8, 220.5). Micro-hematuria was present in approximately two-thirds (68.1%) of patients. Nephrotic range proteinuria and nephrotic syndrome were observed in one-quarter (25.9%) and one-third (31.0%) of the patients, respectively. Approximately half (49.1%) of the patients had acute kidney injury at time of biopsy, while one-fifth (19.8%) had progressive chronic kidney disease. A wide spectrum of histological diagnoses was observed. Glomerulonephritides, tubulointerstitial inflammation and/or glomerular endothelial injuries were diagnosed in 60.8% (48/79) of solid-organ and 80.6% (29/37) of hematological malignancies. Amongst patients with active solid-organ malignancies (n = 20), acute tubulointerstitial nephritis (ATIN) (5/20, 25.0%), IgA nephropathy (3/20; 15.0%) and lupus nephritis (3/20; 15.0%) were the most common primary diagnoses. Amongst patients with active hematological malignancies (n = 17), dysproteinemic kidney disease and/or leukemic infiltration was most frequently diagnosed (6/17; 35.9%). Compared to remission status, active malignancy was not associated with an increased likelihood of an overall diagnosis of glomerulonephritides, tubulointerstitial inflammation and/or glomerular endothelial injuries (p = 0.16). Specifically, the incidence of membranous nephropathy was similar in patients with active malignancy and those in remission (p = 0.42). Amongst patients with HSCT, thrombotic microangiopathy (40.0%) was observed most frequently. Overall, onco-therapeutics-related kidney injuries were present in 13 (11.2%) patients, of which ATIN (38.4%) was most commonly encountered, followed by glomerular endothelial injury (30.7%). Conclusion Kidney biopsy provides valuable diagnostic information amongst cancer patients. While a broad spectrum of histological diagnoses can be observed, glomerular diseases and onco-therapeutics-related kidney injuries remain the most common.

Abstract 4961: Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models

Abstract A large-scale meta-analysis has recently identified Protease-activated receptor 2 (PAR2) gene expression to be significantly associated with resistance to immune checkpoint blockade (ICB) in cancer patients and preclinical models. PAR2 and its ligands (proteases) are indeed upregulated in different cancer types and are expressed by various cells in the tumor microenvironment. In cancer cells, the PAR2 receptor controls cell migration, proliferation, survival, and expression of inflammatory cytokines. In immune cells, it influences the infiltration and phenotype of macrophages and T cells. Therefore, PAR2 represents a promising therapeutic target in oncology and immuno-oncology. A novel series of potent and selective PAR2 inhibitors has been developed at Domain Therapeutics. In vitro experiments demonstrated unique properties of our PAR2 small molecule antagonists when compared to those of competitors. The antagonists inhibit pathogenic signaling pathways (i.e. Gz, G13, Gq, G14 and G15 protein activation as well as intracellular calcium) but not βarrestin2 recruitment, potentially reducing the risks of drug resistance. Furthermore, they maintain high potency and insurmountability in conditions that mimic the tumor microenvironment (high concentration of proteases and acidic pH). Finally, their pharmacokinetic properties are compatible with a once-a-day oral administration and demonstrate no signs of in vivo toxicity except at high doses (&amp;gt;500 mg/kg). Proof-of-concept experiments showed that PAR2 antagonists prevented PAR2-mediated resistance to Gefitinib in vitro and increased the potency of anti-PD1 therapy in vivo in pre-clinical syngeneic mouse models. Immunohistochemistry analyses from cancer patient biopsies confirmed that high expression levels of PAR2 in cancer and stromal cells within the tumor microenvironment significantly associates with the patient overall survival. In conclusion, new potent and selective negative allosteric modulators of PAR2 have been developed, they demonstrate strong potency by alleviating the resistance to both chemo- and immunotherapy in cancer models. These findings confirm the high value of PAR2 as a therapeutic target and demonstrates the relevance of small molecule inhibitors targeting this receptor to treat cancer. Citation Format: Thibaut Brugat, Francesco Bergami, Baptiste Rugeri, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Meriem Semache, Antoine Mousson, Camille Dietsch, Quentin Ruet, Orphée Blanchard, Célia Jacoberger-Foissac, Isabelle Cousineau, Maleck Kadiri, Anne-Laure Blayo, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4961.

A drug repurposing approach for individualized cancer therapy based on transcriptome sequencing and virtual drug screening

RNA-sequencing has been proposed as a valuable technique to develop individualized therapy concepts for cancer patients based on their tumor-specific mutational profiles. Here, we aimed to identify drugs and inhibitors in an individualized therapy-based drug repurposing approach focusing on missense mutations for 35 biopsies of cancer patients. The missense mutations belonged to 9 categories (ABC transporter, apoptosis, angiogenesis, cell cycle, DNA damage, kinase, protease, transcription factor, tumor suppressor). The highest percentages of missense mutations were observed in transcription factor genes. The mutational profiles of all 35 tumors were subjected to hierarchical heatmap clustering. All 7 leukemia biopsies clustered together and were separated from solid tumors. Based on these individual mutation profiles, two strategies for the identification of possible drug candidates were applied: Firstly, virtual screening of FDA-approved drugs based on the protein structures carrying particular missense mutations. Secondly, we mined the Drug Gene Interaction (DGI) database (https://www.dgidb.org/) to identify approved or experimental inhibitors for missense mutated proteins in our dataset of 35 tumors. In conclusion, our approach based on virtual drug screening of FDA-approved drugs and DGI-based inhibitor selection may provide new, individual treatment options for patients with otherwise refractory tumors that do not respond anymore to standard chemotherapy.

Novel EGFR-mutant mouse models of lung adenocarcinoma reveal adaptive immunity requirement for durable osimertinib response

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer patient biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficient nu/nu or Rag1−/− mice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral T cell content and decreased neutrophil content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

EXPRESSION OF ALU REPEAT IN BLOOD PLASMA OF PATIENTS WITH BREAST CANCER DURING NEOADJUVANT CHEMOTHERAPY: AN EXPLORATORY STUDY.

Locally advanced breast cancer(LABC)rates are unusually high in developing countries. There is a need for the identification of predictive biomarkers for the selection of patients who could benefit from neoadjuvant chemotherapy (NAC). As the expression of ALU repeat is increased in cancer and has not been assessed in liquid biopsy of cancer patients, our goal was to assess ALU expression in the blood plasma of LABC patients during NAC. Plasma samples drawn at baseline and at the end of the fourth cycle of chemotherapy were used to determine the plasma levels of ALU-RNA by quantitative real-time PCR. ALU expression from baseline to the fourth cycle of NAC increased from a median relative level of 1870 to 3370 in the whole group (p = 0.03). The increase in ALU-RNA levels in the course of NAC was more pronounced in premenopausal women and in patients with hormone-positive tumors. In patients with complete response to NAC, baseline ALU expression was higher than that in those with partial response. This exploratory study provides evidence that plasma ALU-RNA levels are modulated by the menopausal status and hormone receptor status of breast cancer patients and pre-therapeutic ALU-RNA levels might be useful in predicting the response to chemotherapy in a neoadjuvant setting.

Predictors of Major Hemorrhage After Spleen Core Biopsy in Cancer Patients

In this retrospective study, 232 spleen biopsies from 218 patients with cancer were assessed. Biopsies resulting in hemorrhage requiring hospitalization, transfusion, or other interventions were compared with those that did not. The maximization of the Youden index helped determine the optimal systolic blood pressure (SBP) and platelet count thresholds. There were 15 (7%) major hemorrhages among 211 core biopsies. A multivariate logistic regression model showed that higher SBP, lower platelet count, and the lack of ultrasound guidance were independently associated with major hemorrhage (P < .05). The optimal SBP cutoff was 140 mm Hg, and the platelet count cutoff was 120,000 platelets/μL. In conclusion, the high major hemorrhage rate of 7% among percutaneous core spleen biopsies in patients with cancer may be mitigated by controlling SBP to <140 mm Hg and avoiding biopsy in patients with thrombocytopenia.

Optimising CT-guided biopsies of sclerotic bone lesions in cancer patients

ObjectivesInvestigate the laboratory, imaging and procedural factors that are associated with a tumour-positive and/or NGS-feasible CT-guided sclerotic bone lesion biopsy result in cancer patients.MethodsIn total, 113 CT-guided bone biopsies performed in cancer patients by an interventional radiologist in one institution were retrospectively reviewed. Sixty-five sclerotic bone biopsies were eventually included and routine blood parameters and tumour marker levels were recorded. Non-contrast (NC) biopsy CTs (65), contrast-enhanced CTs (24), and PET/CTs (22) performed within four weeks of biopsy were reviewed; lesion location, diameter, lesion-to-cortex distance, and NC-CT appearance (dense-sclerosis versus mild-sclerosis) were noted. Mean NC-CT, CE-CT HU, and PET SUVmax were derived from biopsy tract and lesion segmentations. Needle diameter, tract length, and number of samples were noted. Comparisons between tumour-positive/negative and next-generation sequencing (NGS)-feasible/non-feasible biopsies determined significant (p < 0.05) laboratory, imaging, and procedural parameter differences.ResultsSeventy-four percent of biopsies were tumour-positive. NGS was feasible in 22/30 prostate cancer patients (73%). Neither laboratory blood parameters, PET/CT availability, size, nor lesion-to-cortex distance affected diagnostic yield or NGS feasibility (p > 0.298). Eighty-seven percent of mildly sclerotic bone (mean 244 HU) biopsies were positive compared with 56% in dense sclerosis (622 HU, p = 0.005) and NC-CT lesion HU was significantly lower in positive biopsies (p = 0.003). A 610 HU threshold yielded 89% PPV for tumour-positive biopsies and a 370 HU threshold 94% PPV for NGS-feasible biopsies. FDG-PET and procedural parameters were non-significant factors (each p > 0.055).ConclusionIn cancer patients with sclerotic bone disease, targeting areas of predominantly mild sclerosis in lower CT-attenuation lesions can improve tumour tissue yield and NGS feasibility.Key Points• Areas of predominantly mild sclerosis should be preferred to areas of predominantly dense sclerosis for CT-guided bone biopsies in cancer patients.• Among sclerotic bone lesions in prostate cancer patients, lesions with a mean HU below 370 should be preferred as biopsy targets to improve NGS feasibility.• Laboratory parameters and procedure related factors may have little implications for CT-guided sclerotic bone biopsy success.

Abstract 1598: Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy characterized by robust, however transient, responses to frontline platinum-based therapy that are rapidly followed by refractory relapses. Treatment resistance is a significant concern in SCLC and a major factor regulating resistance is an emergence of unique cellular populations and an increase in intratumoral heterogeneity (ITH). We hypothesize that these emerging populations expressing various resistance-associated pathways, represent multiple, unique drug-resistant persister cell populations that underlie increased ITH and promote therapeutic resistance and, eventually, relapse, despite the initial response to therapy. Persister cells represent a unique population of cancer cells that are resistant to therapeutic pressure. While most persister cells remain arrested under treatment, a rare sub-population exists that can reverse state and re-enter the cell cycle. We performed single cell RNAseq of relapsed, extensive stage SCLC patient paired core biopsies collected 1) following relapse to standard of care (SOC) platinum chemotherapy plus immune checkpoint blockade (ICB) and 2) after six weeks of further therapy. Unlike what we found in platinum-sensitive disease, there is not a meaningful change in ITH score between cancer cells in the first and second biopsy. Presumably, maximum heterogeneity developed along with relapse to SOC treatment. Biopsies represent SCLC across subtypes (SCLC-A/N/P/I) with a modest loss of transcription factor expression following treatment (e.g., 76.8% to 57.9% NEUROD1). Cancer cells were classified as cycling or non-cycling to identify potential persister cell populations in paired patient core biopsies. Molecular subtype marker ASCL1 was reduced in non-cycling cells, but there was no change in NEUROD1 or POU2F3. Cycling cells demonstrate increased expression of NE genes (SYP, INSM1, UCHL1), biomarkers of response (SLFN11 and CDH1), but also genes associated with resistance (EZH2, NFIB). Non-cycling cells exhibit some common resistance mechanisms (e.g., EMT and MYC, AXL, ZFP36L1, and REST overexpression) and increased expression of inflammatory genes (e.g. HLA family) compared to cycling cells, reminiscent of the SCLC-Inflamed (SCLC-I) subtype. These data suggest that non-cycling persister cells may be more sensitive to ICB, AXL and/or AURK inhibition, while cycling cells may be more responsive to platinum chemotherapy, epigenetic modifiers or DNA repair targeted therapies. Clinically, these data underscore the importance of maximizing and maintaining the initial response in platinum-sensitive SCLC tumors, while highlighting the transcriptional complexity underlying SCLC’s profound treatment resistance following SOC and address the major need to develop combination therapies to target these distinct cell populations. Citation Format: C. Allison Stewart, Yuanxin Xi, Runsheng Wang, Michael M. Frumovitz, Jing Wang, Lauren A. Byers, Carl M. Gay. Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1598.

Abstract 4246: ABL103, A novel T-cell engaging bispecific antibody, exhibits potent in vitro and vivo antitumor activity and low toxicity via B7-H4 dependent 4-1BB activation in tumor microenvironment

Abstract B7-H4 (B7x, VTCN1), a member of the B7-family, is overexpressed in majority of cancer patients with ovarian, endometrial and breast cancers. B7-H4 expression was also observed in tumor associated macrophages and implicated as an immune checkpoint regulating T cell responses. Although B7-H4 expression in normal tissues is quite limited, tumor-specific immune response by ABL103 would minimize potential adverse effects. ABL103, B7-H4 Grabody-T, is a First-in Class bispecific antibody targeting B7-H4 and 4-1BB and augments T cell function by a dual mechanism, i.e., 1) blocks the B7-H4 mediated T cell inhibition, and 2) elicits superior T cell activation through B7-H4-dependent 4-1BB clustering. Our data showed that simultaneous binding of B7-H4 and 4-1BB by ABL103 led to potent in vitro T cell activation only in the presence of B7-H4 expressing tumor cells. In the established tumor model, ABL103 potently inhibited tumor progression in a dose-dependent manner and showed higher rate of complete remission (CR) at 2 and 10 mg/kg dose groups. Moreover, mice were protected from the tumor re-challenge 3 months after cessation of ABL103 treatment, suggesting long-term memory has been established. In 4-week pilot tox study using cynomolgus monkeys, ABL103 was tolerable up to 100 mg/kg dosed weekly with no ABL103-related toxicity observations. To understand the relationship of target expression, expression of B7-H4, CD4, CD8, PD-L1, and 4-1BB was examined in 142 ovarian cancer patient biopsies. About 83% of ovarian cancer patient tissues showed B7-H4 positive staining and 62% of them showed strong positive. In addition, the staining of 4-1BB as well as CD4 and CD8 T cells were observed in both tumor nest and stroma, suggesting that cross-linking of B7-H4 and 4-1BB is feasible in tumor microenvironment. Overall, ABL103 has a strong in vitro and vivo anti-tumor activity and good safety profile via B7-H4-dependent 4-1BB activation. This strongly suggests ABL103 is a promising therapeutic agent potentially benefitting patients with B7-H4 overexpression. Citation Format: Kyungjin Park, Yangsoon Lee, Saeyi Lim, Kyeongsu Park, Eunjung Kim, Hanbyul Lee, Jiseon Yoo, Youngdon Pak, Yeunju Kim, Minji Ko, Jonghwa Won. ABL103, A novel T-cell engaging bispecific antibody, exhibits potent in vitro and vivo antitumor activity and low toxicity via B7-H4 dependent 4-1BB activation in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4246.

Evaluation of pharmacodynamic and patient enrichment biomarkers for SAR444881, a first-in-class anti-ILT2 monoclonal antibody for cancer immunotherapy.

2571 Background: Leukocyte Ig-like receptor B1 [LILRB1; ILT2] is an inhibitory receptor expressed on various immune cells. ILT2 binds to classical and nonclassical MHC class I molecules, with highest affinity to HLA-G. ILT2-mediated inhibition leads to impairment of immune cell proliferation, differentiation, phagocytosis, cytotoxicity and cytokine secretion. Antagonism of ILT2 signaling may serve as a novel target for anti-cancer immunotherapy. SAR444881 (BND-22) is a novel humanized IgG4 monoclonal antagonist antibody which selectively binds to ILT2 and blocks its interaction with MHC I molecules. SAR444881 induces robust macrophage and lymphocyte-driven anti-tumor activity in in vitro and in vivo models. To overcome the limitation that LILRB proteins are not expressed in rodents, we conducted a series of in vivo studies using humanized mouse models, cancer patient biopsies and ex vivo co-culture systems to interrogate the pharmacodynamic (PD) response of ILT2 antagonism as well as inform the combinatorial and patient enrichment strategies for SAR444881. Methods: SAR444881-induced modulation of PD biomarkers was evaluated in humanized xenograft models. Ex vivo co-culture system has been established using patient tumor tissues and isolated PBMC or other immune cells. Biomarker expression on immune cells and secreted soluble proteins were monitored by flow cytometry and ELISA, respectively. Procured tumor samples of patients with various advanced solid tumors, including Head and neck squamous cell carcinoma (HNSCC), Gastric Cancer (GC), Non-Small Cell Lung Cancer (NSCLC) and Colorectal Cancer (CRC) were utilized to evaluate protein or gene expression levels of potential predictive biomarkers (ILT2, HLA = G, PD-L1) using immunohistochemistry (IHC) and whole transcriptomic analysis (RNAseq). Results: 1) PD biomarkers: in murine models, SAR444881 modulates intra-tumoral sub-populations of CD8+ T cells, NK cells and Macrophage polarization; 2) Combination strategy: functional study in an ex vivo system showed that addition of an anti-PD-1 antibody induced an increased production of pro-inflammatory cytokines, including IFNγ and TNFα, compared to SAR444881 or anti-PD-1 alone. Concomitantly, combining SAR444881 with cetuximab resulted in increased phagocytosis compared to isotype control. 3) Predictive biomarkers: protein and gene profiling results suggest enriched expression of ILT2 in majority of biopsies from several tumor types including NSCLC, HNSCC and CRC. Conclusions: These data inform the PD response biomarkers, combination, and patient enrichment strategies for the clinical development of SAR444881 to maximize its benefits for cancer patients. An ongoing phase 1/2 trial of SAR444881 mono- and combination therapy in patients with advanced solid tumors is testing these concepts in the clinic (NCT04717375).

Clinical involvement of Dickkopf protein‐related protein 3 (DKK3) and Prolyloligopeptidase with tumor progression and metastasis onset of Glioblastoma multiforme

Glioma are common malignant brain tumors, among which glioblastoma multiforme (GBM) has the worst prognosis. GBM is characterized by diffuse infiltration of the brain tissue surrounding the bulk of the tumor, associated with chromosomal and genetic mutations that determine the uncontrolled growth of brain cells and tumor cell chemoresistance. Glioblastomas only rarely metastasize to sites outside the central nervous system, for reasons that are poorly understood, however, recent investigations of other tumor types have suggested that particular molecular features may correlate with metastatic potential such as the activation of many angiogenic factors. Aberrant Wnt signaling has been described as a key player in the initiation of and/or maintenance and development of glioma and Dickkopf protein‐related protein 3 (Dkk‐3), as a tumor suppressor, represents the most promising modulators of the Wnt pathway. Moreover, prolyl‐oligopeptidase (PREP) is a serine protease involved in the angiogenesis and inflammatory process and many research studies highlighted the role of this protein in the metastasis spread.The aim of the present study was to evaluate the possible correlation between Dkk‐3 expression and different GBM patients’ parameters in the regulation of tumor growth processes, by investigating the possible role of PREP in GBM‐related metastasis.Biopsies of GBM and GBM‐related metastasis were taken from patients operated at the Neurosurgery of the Polyclinic “G. Martino” of Messina. The study included 20 cases of GBM of different histological grading, taken by female and male patients, aged between 43 to 77 years. The statistical analysis was performed correlating the extent of Dkk‐3 expression of the single GMB samples with different patient parameters: gender, age, survival and % Ki‐67 labeling index as well the evaluation of PREP modulation in GBM‐related metastasis.Clinical data showed that the down‐regulation of Dkk‐3 was correlated with survival and age; specifically, a low expression of Dkk‐3 tends to be higher in male patients compared to female patients, with an age media of 57 years. Instead, the analysis between Dkk‐3 expression and survival after the first GBM surgery did not show a statistically significant correlation. Also, increased reactivity for Ki67 and ATRX was found in biopsies of patients with Dkk‐3 overexpression, associated with an incidence of the mutation in the gene encoding the isocitrate dehydrogenase IDH132. Moreover, a significant PREP expression was found in both GBM primary tumor and GBM‐related metastasis.In conclusion, the overexpression of Dkk‐3 on GBM samples showed a significant reduction of these markers in biopsies of cancer patients compared to controls, while PREP modulation could slow down metastatic expansion by reducing angiogenesis, suggesting new possible molecular targets for future therapeutic protocols.

Clinical application of liquid biopsy in cancer patients

BackgroundThis study was to determine the prevalence and clinical significance of clonal hematopoiesis (CH)-related variants, and somatic and germline mutations in cancer patients and healthy individuals.MethodsWe performed next-generation sequencing of 275 cancer-related genes be-tween plasma and white blood cells in 92 cancer patients and 47 controls without cancer. Blood samples were recruited from May 2017 to July 2021, and blood cancer patients were excluded. For all statistical analysis in this study, p < 0.05 was considered statistically significant.ResultsOverall, 38.04% of patients and 46.81% of controls harbored at least one CH-related mutation in plasma cell-free DNA. Based on our results, older cancer patients exhibited a CH phenomenon more frequently than younger patients (p = 0.0024). A total of 39 somatic pathogenic (P)/likely pathogenic (LP) mutations were identified in 17 genes in 21 of 92 patients. We found that the presence of P/LP variants in cancer-related gene predicted shorter overall survival (OS) (p = 0.001). Multivariate analysis adjusted for CH-related mutations, germline mutations, and tumor stage, also indicated that somatic mutations correlated significantly with OS (p = 0.022). Moreover, the frequency of a germline P/LP variant was that of seven of 92 individuals in the cancer group and one of 42 individuals in the control group.ConclusionsWe characterized the CH-related variants, and somatic and germline mutations in cancer patients and healthy individuals, and the results have important clinical significance.

In situ detection of single microRNA molecules by transient hybridization utilizing FRET-based probes

MicroRNAs are highly implicated in an abundance of cellular pathways relevant to human health and disease, including cancer. The critical role miRNAs play in gene regulation and biological function, underscored in their dysregulation in tumor cells and presence as biomarkers in biopsies of cancer patients, warrants further investigation. Current techniques utilized for quantification of miRNAs usually lack adequate sensitivity and quantitative accuracy for rare or hard-to-amplify species. To overcome these pitfalls, we are developing a miRNA single-molecule imaging method for in situ detection by FRET-based probes.