Fresh Tumor Tissue Research Articles

LINC00365 as a potential biomarker for total nephrectomy in advanced-stage renal cell carcinoma patients

BackgroundRenal cell carcinoma (RCC) is one of the most frequent urological cancers globally that has a good prognosis in the early tumor stages. However, there is a poor prognosis in metastatic RCC patients. Therefore, it is needed to evaluate the molecular biology of RCC progression to introduce the efficient diagnostic and therapeutic markers in these patients. Long non-coding RNAs (lncRNAs) have key roles in regulation of molecular mechanisms during RCC progression. In the present study, we assessed the levels of LINC00365 expressions in RCC patients to suggest that as a tumor marker in these patients. MethodsFifty fresh RCC tumor tissues and their normal margins were collected to assess the levels of LINC00365 expressions and probable correlations with clinicopathological features of RCC patients. ResultsThere was significant LINC00365 up regulation in females compared with males (p=0.050). Among the RCC patients with total nephrectomy, there was a significant LINC00365 up regulation in advanced stage compared with primary stage tumors (p=0.035). RCC patients older than 60 years old who were undergone the total nephrectomy had also significant LINC00365 up regulation compared with RCC patients younger than 60 years old (p=0.039). Conclusionsgiven the significant increase in LINC00365 expression in advanced stage RCC tumors and patients over 60 years old who had total nephrectomy; it could serve as a useful diagnostic marker in screening programs for old high-risk individuals. It was also noticed that female RCC patients had elevated levels of LINC00365 expressions in their tumor samples, suggesting its potential use as a gender-specific diagnostic marker for high-risk females. Nevertheless, evaluating the levels of LINC00365 in serum samples of RCC patients is necessary to suggest that as a reliable diagnostic marker in clinical settings.

Odontogenic Myxomas Harbor Recurrent Copy Number Alterations and a Distinct Methylation Signature.

Odontogenic myxoma is a rare, benign, and locally aggressive tumor that develops in the tooth-bearing areas of the jaw. The molecular mechanisms underlying odontogenic myxomas are unknown and no diagnostic markers are available to date. The aim of this study was to analyze DNA methylation and copy number variations in odontogenic myxomas to identify new molecular signatures for diagnostic decision-making. We collected a cohort of 16 odontogenic myxomas from 2006 to 2021 located in the mandible (n = 10) and maxilla (n = 6) with available formalin-fixed paraffin-embedded or fresh frozen tumor tissue from a biopsy or resection material. Genome-wide DNA methylation and copy number variation data were generated from 12 odontogenic myxomas using the Illumina Infinium Methylation EPIC array, interrogating >850,000 CpG sites. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) revealed that odontogenic myxomas formed a distinct DNA methylation class. Copy number profiling showed recurrent whole-chromosome gains (trisomies) of chromosomes 5, 8, and 20 in all cases, and of chromosomes 10, 12, and 17 in all except one case. In conclusion, odontogenic myxomas harbor recurrent copy number patterns and a distinct DNA methylation profile, which can be used as an additional diagnostic tool in the appropriate clinical and radiologic context. Further research is needed to explain the genetic mechanisms caused by these alterations that drive these locally aggressive neoplasms.

Claudin 7 suppresses invasion and metastasis through repression of a smooth muscle actin program.

Metastasis initiates when cancer cells escape from the primary tumor, which requires changes to intercellular junctions. Claudins are transmembrane proteins that form the tight junction, and their expression is reduced in aggressive breast tumors. However, claudins' roles during breast cancer metastasis remain unclear. We used gain- and loss-of-function genetics in organoids isolated from murine breast cancer models to establish that Cldn7 suppresses invasion and metastasis. Transcriptomic analysis revealed that Cldn7 knockdown induced smooth muscle actin (SMA)-related genes and a broader mesenchymal phenotype. We validated our results in human cell lines, fresh human tumor tissue, bulk RNA-seq, and public single-cell RNA-seq data. We consistently observed an inverse relationship between Cldn7 expression and expression of SMA-related genes. Furthermore, knockdown and overexpression of SMA-related genes demonstrated that they promote breast cancer invasion. Our data reveal that Cldn7 suppresses breast cancer invasion and metastasis through negative regulation of SMA-related and mesenchymal gene expression.

CD19+CD73+ B cells infiltration indicates poor prognosis and unfavorable responses to immunotherapy in gastric cancer

ObjectivesCluster of Differentiation 73 (CD73) is expressed on immune cells and plays a significant role in tumor inhibition by suppressing antitumor immunity. The objectives of this study were to explore the expression and functional mechanisms of CD73 on B cells in patients with gastric cancer (GC). MethodsThe prognostic significance of CD19+CD73+ B cells was evaluated in 390 GC patients through dual immunohistochemistry staining. Flow cytometry was employed to analyze the phenotype of the CD19 subpopulation using fresh tumor and non-tumor tissue samples from 8 GC patients. A bioinformatics analysis of CD19+CD73+ B cells was also performed within the scRNA-seq cohort, and the CD19+ B cell subtype was assessed using multiple immunofluorescence staining. ResultsThe infiltration of CD19+CD73+ B cells was observed to be elevated in gastric cancer (GC) tissue compared to normal tissues. A strong correlation was observed between high CD19+CD73+ B cell infiltration, poor overall survival, and diminished responsiveness to neoadjuvant immunotherapy in GC. These cells emerged as a novel subset of regulatory B cells (Bregs) linked to adenosine metabolism and the exhaustion of CD8+ T cells. The CD19+CD73+ B cells also correlated with the production of immunosuppressive cytokines IL-10 and TGFB1. Further analysis indicated an association between CD19+CD73+ B cells and advanced-stage GC. ConclusionsThe presence of CD19+CD73+ B cells in GC may serve as a prognostic indicator for clinical outcomes and a predictive marker for poor responsiveness to neoadjuvant immunotherapy. The correlation between the presence of CD19+CD73+ B cells and CD8+ T cell exhaustion, along with immunosuppression, highlights the tumor-promoting function of these cells.

Helicobacter pylori Infection Affects the Tumor Immune Microenvironment of Esophageal Cancer Patients.

We herein examined T cell immunity in esophageal cancer patients with and without Helicobacter pylori infection to establish a foundation for immunotherapeutic strategies targeting esophageal cancer in the presence of H. pylori infection. Twenty-six patients with esophageal squamous cell carcinoma between 2015 and 2017 were enrolled in the present study. Serum antibodies against H. pylori were measured. Fresh tumor tissues were obtained by endoscopic biopsy or from surgical resection. A cell suspension of these tissues was subjected to a flow cytometric analysis. Among the 26 patients analyzed, 10 (38.5%) were seropositive for H. pylori. The flow cytometric analysis of tumor-infiltrating lymphocytes revealed that the percentage of CD103+CD4+ T cells in esophageal tumors was significantly lower in H. pylori-positive patients than in H. pylori-negative patients (p=0.0105). Conversely, the percentage of CD45RA-CD25hi effector Treg cells in esophageal tumors was significantly higher in H. pylori-positive patients than in H. pylori-negative patients (p=0.0022), indicating an immunosuppressive tumor microenvironment in the former. Following neoadjuvant chemotherapy, the number of CD45RA-CD25hi effector Treg cells decreased (p=0.0248). The tumor immune microenvironment of esophageal cancer patients with H. pylori infection exhibited an immunosuppressive phenotype. The targeting of Treg cells has potential in immunotherapy for this patient population.

Injectable and Sprayable Fluorescent Nanoprobe for Rapid Real-Time Detection of Human Colorectal Tumors.

The development of minimally invasive surgery has greatly advanced precision tumor surgery, but sometime suffers from restricted visualization of the surgical field, especially during the removal of abdominal tumors. A 3-D inspection of tumors could be achieved by intravenously injecting tumor-selective fluorescent probes, whereas most of which are unable to instantly distinguish tumors via in situ spraying, which is urgently needed in the process of surgery in a convenient manner. In this study, this work has designed an injectable and sprayable fluorescent nanoprobe, termed Poly-g-BAT, to realize rapid tumor imaging in freshly dissected human colorectal tumors and animal models. Mechanistically, the incorporation of γ-glutamyl group facilitates the rapid internalization of Poly-g-BAT, and these internalized nanoprobes can be subsequently activated by intracellular NAD(P)H: quinone oxidoreductase-1 to release near-infrared fluorophores. As a result, Poly-g-BAT can achieve a superior tumor-to-normal ratio (TNR) up to 12.3 and enable a fast visualization (3min after in situ spraying) of tumor boundaries in the xenograft tumor models, Apcmin/+ mice models and fresh human tumor tissues. In addition, Poly-g-BAT is capable of identifying minimal premalignant lesions via intravenous injection.

Abstract A016: Companion Diagnostics (CDx) to identify hallmarks of Alternative Lengthening of Telomeres (ALT)

Abstract Maintenance of telomeres in the absence of telomerase is known as Alternative Lengthening of Telomeres (ALT), which is prevalent in 10-15% of all cancers. Tessellate BIO targets ALT mechanisms to drive cell death by synthetic lethality. To identify patients who will benefit from our therapy, we are developing companion diagnostics (CDx). In this study, established hallmarks of ALT are investigated as CDx target in tissue-derived samples. In fresh and FFPE tumor tissue, presence and abundance of different ALT hallmarks were measured, including RPA phosphorylation, C-circles and telomeric single stranded DNA. While not all markers pass the stringent requirements of a CDx, C-circles prove to be a reliable biomarker. To measure C-circles, a robust and scalable PCR-based protocol was developed, facilitating easy implementation in the clinic. Utilizing this protocol, C-circles were successfully measured across different tissue types. Further, the protocol allowed for C-circle quantification in cell lines, showcasing its utility across matrices of varying complexity. Successful development of the C-circle CDx enables patients selection based on ALT mechanisms herewith improving the response rate of targeted therapies in patients and accelerating new discoveries in the ALT space. Citation Format: Ganesh Kadamur, Dalia Tarantino, Freddie Grogono, Patrick Von Morgen, Salvina Tammaccaro, Diana Zatreanu, Donata Federici Canova, Jana Wolf, Corne van den Kieboom, Katie Chapman, Joris Schuurmans, Jurgen Moll. Companion Diagnostics (CDx) to identify hallmarks of Alternative Lengthening of Telomeres (ALT) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A016.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PA) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system to establish organoid models from parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, cytokeratin 7, and epithelial membrane antigen in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusions: This study successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

A phase IV trial to confirm the efficacy of olaparib in combination with bevacizumab as maintenance frontline treatment of HRD-positive ovarian tumours (IOlanTHe).

TPS5628 Background: PAOLA-1/ENGOT-ov25 trial demonstrated that Olaparib (O) plus Bevacizumab (B) determined a significant survival benefit in patients (pts) with advanced, high-grade, epithelial ovarian, peritoneal and/or fallopian-tube cancer (HEOC) presenting defects of homologous recombination repair system (HRD+) in clinical response after first-line platinum-based chemotherapy (CT) plus B. Methods: IOLANTHE is a multicenter Italian phase IV trial (sponsored by the MaNGO group, Protocol Number IRFMN-OVA-8542) aiming to confirm in a setting close to clinical practice the efficacy of this combination in pts with HRD+HEOC. As the study includes a consistent translational component, all suspicious advanced cases will be considered for study enrollment. Pts with a confirmed pathological diagnosis of HEOC will be included in the step 1 of the trial which main inclusion criteria are the following: -availability of formalin-fixed, paraffin-embedded samples for the central testing of HRD status (performed by Myriad Mychoice CDx Plus assay) - suitability to receive CT + B. Only HRD+ pts responding to first line CT plus at least one cycle of B will be enrolled in the step 2 of the trial and will be treated with B 15 mg/kg every 3 weeks plus O 300 mg twice daily. Considering the PAOLA-1 median progression free survival (PFS) of 37.2 months (mo) in the B-O arm, with a sample size of 90 pts followed for 24 months and setting a one-side error of 5%, we will have a statistical power of 80% to demonstrate a PFS-24mo≥64% with an upper critical value of 61%. In order to have 90 patients eligible for the step 2 of the study, 190 pts are needed to be enrolled in the step 1 of the clinical study (as approximately 80% of pts with HEOC will respond to CT and out of these, 60% will be HRD+). The translational subproject 1 will consist of the analysis of circulating-tumor (ctDNA), using whole genome sequencing aiming - to correlate residual tumour after surgery and ctDNA levels - to anticipate the diagnosis of progression - to monitor the mutational status of HR-related genes and other genes such as Tp53BP1, POLQ, REV7 probably involved in PARPi resistance during the maintenance therapy. The translational subproject 2 will be developed to compare pts’ response to therapy with that of cancer cells, tested by organotypic model treated with the combination. These models will be generated using fresh tumor tissue and ascitic fluid for the isolation of the tumor cells and macroscopically healthy omentum for the isolation of the mesothelial cells and fibroblasts. Eight centers have been activated and other 6 are waiting for the activation with 19 patients registered and 3 screening failures. The enrollment is continuing, according to protocol timeline (12 months for enrollment, 6 months for surgery and CT and 24 months for maintenance treatment). Clinical trial information: NCT06121401 .

Baseline heterogeneity of myeloid and stroma cells to predict pCR, baseline lymph node (LN) metastasis, and 1-year EFS in locally advanced ESCC with neoadjuvant immunotherapy: Final results from a single-arm, phase II clinical trial (SEEK-01).

e14661 Background: Response of neoadjuvant immunotherapy can vary among patients in locally advanced ESCC, with the lack of a promising biomarker which need fully capture the heterogeneity of TME. Now, based on final results of the SEEK-01 study (2023 ASCO, P#4047), we have, for the first time, established a precise correlation from baseline TME to pathological response, baseline LN status, and 1-year EFS. Methods: In SEEK-01 study, 22 locally advanced ESCC patients (cT2-4NxM0) were enrolled. Following neoadjuvant chemotherapy (paclitaxel-albumin+carboplatin) combined with tislelizumab, patients underwent minimal invasive esophagectomy (MIE). Fresh tumor tissues were obtained via endoscopy prior to neoadjuvant therapy for scRNAseq analysis. Pathological response was evaluated using the irRVT score, leading to classification into pCR (irRVT=0%), MPR (0<irRVT<10%), and non-responder (irRVT>60%). Baseline LN status was determined based on pathological diagnosis after surgery. We identified subclusters of tumor-associated myeloid cells and stroma cells that significantly predicted pathological response, baseline LN metastasis and recurrence within 1 year. Prediction model of efficacy for neoadjuvant immunotherapy was developed using baseline expression ratios of differential subclusters. Results: Among 22 enrolled patients, 18 completed neoadjuvant therapy and MIE. 4 achieved pCR, 2 got MPR and 6 were non-responder. 12 of 18 proved baseline LN metastasis. 5 of 18 suffered recurrence within 1 year. Myeloid cells, cancer-associated fibroblast (Caf) and endothelium (Endo) shew heterogeneity that can predict pathological response, baseline LN metastasis or recurrence within 1 year. Upgrade of CXCL9+ tumor-associated-macrophage (TAM), LAMP3+mregDC3 was a strong predictor of pCR, while the upgrade of STAT1+TAM, C1q+TAM, IL1b+TAM, IL1b+ Neutrophil, C1q+ Neutrophil was a solid biomarker of non-responder. Besides, presence of PTGS1+Caf, RGS5+Caf, S100B+Caf, RND1+Endo and S100+Endo highly correlated with baseline LN metastasis and recurrence within 1 year, which was better than PET-CT. On this basis, we constructed predictive models for pathological response. With further validated by public scRNAseq and TCGA database, it showed excellent predictive ability and robustness. Conclusions: The final results of the SEEK-01 study demonstrate the differential baseline infiltration of myeloid cells that can predict pathological response of neoadjuvant immunotherapy. The characteristic changes in Caf and Endo in TME can predict occult LN metastasis and 1-year EFS. A predictive model for efficacy of neoadjuvant immunotherapy in locally advanced ESCC was developed to facilitate individualized and precise neoadjuvant therapy before treatment. Clinical trial information: NCT05807542 .

Cervical cancer-specific long non-coding RNA landscape reveals the favorable prognosis predictive performance of an ion-channel-related signature model.

Ion channels play an important role in tumorigenesis and progression of cervical cancer. Multiple long non-coding RNA genes are widely involved in ion channel-related signaling regulation. However, the association and potential clinical application of lncRNAs in the prognosis of cervical cancer are still poorly explored. Thirteen patients with cervical cancer were enrolled in current study. Whole transcriptome (involving both mRNAs and lncRNAs) sequencing was performed on fresh tumor and adjacent normal tissues that were surgically resected from patients. A comprehensive cervical cancer-specific lncRNA landscape was obtained by our custom pipeline. Then, a prognostic scoring model of ion-channel-related lncRNAs was established by regression algorithms. The performance of the predictive model as well as its association with the clinical characteristics and tumor microenvironment (TME) status were further evaluated. To comprehensively identify cervical cancer-specific lncRNAs, we sequenced 26 samples of cervical cancer patients and integrated the transcriptomic results. We built a custom analysis pipeline to improve the accuracy of lncRNA identification and functional annotation and obtained 18,482 novel lncRNAs in cervical cancer. Then, 159 ion channel- and tumorigenesis-related (ICTR-) lncRNAs were identified. Based on nine ICTR-lncRNAs, we also established a prognostic scoring model and validated its accuracy and robustness in assessing the prognosis of patients with cervical cancer. Besides, the TME was characterized, and we found that B cells, activated CD8+ T, and tertiary lymphoid structures were significantly associated with ICTR-lncRNAs signature scores. We provided a thorough landscape of cervical cancer-specific lncRNAs. Through integrative analyses, we identified ion-channel-related lncRNAs and established a predictive model for assessing the prognosis of patients with cervical cancer. Meanwhile, we characterized its association with TME status. This study improved our knowledge of the prominent roles of lncRNAs in regulating ion channel in cervical cancer.

Tislelizumab plus concurrent chemoradiotherapy versus concurrent chemoradiotherapy for elderly patients with inoperable locally advanced esophageal squamous cell carcinoma: A multicenter, randomized, parallel-controlled, phase II clinical trial.

TPS4183 Background: There is few high-level prospective randomized studies and international consensus guideline on treatment in elderly patients with inoperable locally advanced esophageal squamous cell carcinoma (LAESCC). Double-agent intravenous chemotherapy concurrent with radiotherapy is the standard of care for LAESCC. However, it’s based on clinical trials that only enrolled patients ≤ 75 years old and infeasible in elderly patients. Oral S-1 plus concurrent radiotherapy for elderly ESCC showed tolerable toxicity and survival benefit compared with radiotherapy alone. While, the 3-year overall survival (OS) rate is limited about 40%. Therefore, it urgently needs find new treatment strategies to further improve the efficacy in elderly ESCC patients. Tislelizumab, the antii-PD-1 antibody, has recently been proved in the first- and second-line standard treatment for advanced ESCC. It shows mild toxicities and might be well-tolerated in the elderly. Here, we conducted the first clinical trial to assess the efficacy and safety of tislelizumab plus concurrent chemotherapy (CCRT) versus CCRT for elderly patients with inoperable LAESCC. Methods: The study is an open label, multicenter, parallel-controlled, investigator-initiated phase II clinical trial in elderly patients with inoperable LAESCC. We plan to enroll 136 elderly patients with LAESCC who will be randomly divided into two groups: tislelizumab combined with CCRT group (tislelizumab + radiotherapy + S-1) and CCRT group (radiotherapy + S-1). Tislelizumab is given intravenously on the first day of CCRT at the dose of 200 mg once every 3 weeks (Q3W). Radiotherapy is delivered at 50.4 Gy/1.8 Gy/28 fraction (5 times per week). S-1 is orally given at the dose of 40-60 mg from day1 to day14 and day 22 to 35. We will evaluate the efficacy and safety during the treatment and follow-up period until disease progression, death or the end of the trial. Considering the median progression free survival (PFS) is 16 months in elder receiving CCRT in history studies, we expect the median PFS would be 28 months at the hazard ratio of 0.57. The final sample size consists in 136 patients by 1:1 randomized in the two groups with 80% power and one-sided alpha and calculating for a 10% dropout rate. The primary endpoint is PFS. Secondary endpoints include OS, objective response rate, duration of objective response and severity of adverse events. Fresh or archival tumor tissues and peripheral blood samples will be used in the exploratory studies. This trial started on October 10, 2023 and has enrolled 19 patients till February 1, 2024. Clinical trial information: NCT06061146 .

ICARUS-LUNG01: A phase 2 study of datopotomab deruxtecan (Dato-DXd) in patients with previously treated advanced non-small cell lung cancer (NSCLC), with sequential tissue biopsies and biomarkers analysis to predict treatment outcome.

8501 Background: Few treatments with limited benefit are currently available after failure of targeted therapies, immunotherapy and platinum-based chemotherapy in patients (pts) with advanced NSCLC. Dato-DXd is an antibody-drug conjugate (ADC) composed of a TROP-2-directed monoclonal antibody linked to a topoisomerase I inhibitor via a peptide cleavable linker. In the phase 3 TROPION-Lung01 study, Dato-DXd demonstrated a statistically significant improvement of PFS over docetaxel in previously treated pts with advanced NSCLC. Here we report the results of ICARUS-Lung01 (NCT04940325), a multi-center, single-arm, phase 2 study evaluating activity, safety, and biomarkers of response/resistance to Dato-DXd in pretreated pts with advanced NSCLC. Methods: Intravenous Dato-DXd 6 mg/kg was given every 21 days to pts with advanced NSCLC, ECOG 0/1, who progressed on 1-3 lines of therapy (including actionable genomic alteration (AGA)-specific therapy if indicated). All pts underwent fresh tumor tissue biopsies at baseline, on-treatment (week 3 or 6) and end of treatment. Primary endpoint: investigator-assessed confirmed objective response rate (ORR). A set of translational analyses was performed to determine biomarkers associated with response/resistance, including TROP2 tumor membrane expression, TROP2-dynamics and spatial distribution (by AI-digital pathology), genomics, transcriptomic, spatial proteomics (by imaging mass cytometry) and CTCs. Results: A total of 100 pts received ≥1 dose: median age 60 years (26-83); 62% males, 89% smokers, 82% non-squamous (NSQ) histology, 23% AGA, median number of prior therapy lines 2 (1-5). At data cut-off (Dec 04, 2023), 92% discontinued therapy, 92 % had disease event, 67% died. Median treatment duration was 2.8 months (mos) (95%CI, 2.1-4.8), median follow-up 19.4 mos (95%CI, 18.2-20.4). Efficacy and safety results are shown in the table. Conclusions: In this heavily pretreated population, Dato-DXd showed similar efficacy and safety results to those reported in TROPION-Lung01. Patients with NSQ appeared to derive the greatest benefit. Translational analyses to determine biomarkers associated with response and/or resistance will be presented. Clinical trial information: NCT04940325 . [Table: see text]

Abstract PO4-18-11: Phase 1 study of the tissue factor-targeting antibody-drug conjugate XB002 in patients with advanced solid tumors (JEWEL-101): Design of the triple-negative and hormone receptor-positive breast cancer expansion cohorts

Abstract Background: Tissue factor (TF) is a transmembrane protein that functions as a factor VIIa receptor, initiating the extrinsic coagulation cascade. TF is aberrantly expressed in multiple solid tumors, including breast cancer (BC), and its expression is associated with disease progression and poor prognosis (van den Berg et al. Blood 2012). Microtubule-targeting agents are a cornerstone of treatment for metastatic BC. XB002 is a novel antibody-drug conjugate (ADC) composed of a high-affinity TF-directed human monoclonal antibody conjugated to the zovodotin linker-payload. Zovodotin consists of an auristatin-based payload and a protease-cleavable linker and is designed to lower off-target deconjugation and improve tolerability compared with other microtubule inhibitor linker-payloads. In preclinical studies, XB002 did not interfere with coagulation and demonstrated antitumor activity with an encouraging safety profile. XB002 is being evaluated in JEWEL-101, an ongoing trial in patients (pts) with advanced solid tumors, including BC. Preliminary results from the dose-escalation stage of JEWEL-101 showed XB002 was well tolerated at multiple dose levels with no bleeding events or treatment-related peripheral neuropathy (PN) and low-grade ocular toxicity (Ulahannan et al. ENA 2022). Presented here is the study design of the tumor-specific expansion stage, including triple-negative BC (TNBC) and hormone receptor-positive BC (HR+ BC) cohorts. Trial design and statistical methods: JEWEL-101 (NCT04925284) is a multicenter, open-label, phase 1 trial evaluating the safety, tolerability, pharmacokinetics (PK), and antitumor activity of XB002 in advanced solid tumors. Single-agent XB002 at the recommended dose will be assessed in the BC expansion cohorts utilizing Simon’s Two-Stage design. Eligibility criteria: The trial will enroll adult pts with TNBC (ER-/PR-/HER-2-negative or low) and HR+ BC (ER+ and/or PR+ and HER-2-negative or low) into 2 separate cohorts. HER-2 negativity is defined as either HER-2 negative or low by local ISH or IHC assessment. For both cohorts, pts must have received 1–3 lines of prior systemic anticancer therapies for locally advanced or metastatic disease. Pts in the HR+ BC cohort must have received prior endocrine therapy and CDK4/6 inhibitor. All pts must have cytologically or histologically and radiologically confirmed BC that is inoperable, locally advanced, metastatic, or recurrent. Documented radiographic progression during or following their last systemic anticancer therapy is required. All pts must also have measurable disease per RECIST 1.1 by investigator assessment, archival (< 2 years) or fresh tumor tissue, ECOG PS 0 or 1, and adequate organ and marrow function. Pts with grade 1 PN are allowed. Concomitant anticoagulation therapy while receiving XB002 is permitted if clinically indicated. Key exclusions are prior use of TF-targeting or auristatin derivate-based ADC; uncontrolled, significant intercurrent or recent illness, including acute or chronic significant ocular disorders; major surgery within 4 weeks; and corrected QT interval calculated by the Fridericia formula >480 ms per ECG. Specific aims: The primary objective of the expansion stage is to evaluate the preliminary efficacy of XB002 by estimating the objective response rate per RECIST 1.1 as assessed by the investigator. Secondary objectives include safety and tolerability, PK, immunogenicity, progression-free survival, duration of response, and overall survival. Accrual: Up to 28 pts will be enrolled into each BC cohort. Enrollment is planned across sites in the US, Europe, and Asia-Pacific. Contact information: Exelixis Clinical Trials, druginfo@exelixis.com, 1-888-393-5494 Citation Format: Susanna Ulahannan, Melissa Johnson, Mia Weiss, Andrae Vandross, Sofia Vidal-Cardenas, Mustafa Syed, Anthony Tolcher. Phase 1 study of the tissue factor-targeting antibody-drug conjugate XB002 in patients with advanced solid tumors (JEWEL-101): Design of the triple-negative and hormone receptor-positive breast cancer expansion cohorts [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-11.

Denosumab Induces Neoplastic Stromal Cell Apoptosis Via p62 Downregulation Dependent on Autophagy Pathway in Giant Cell Tumour of Bone.

As the only humanized monoclonal antibody against receptor activator of nuclear factor-κB ligand (RANKL) for giant cell tumour of bone (GCTB) therapy, denosumab has limited antitumour effect on neoplastic stromal cells. Nevertheless, its mechanism of action has not yet been clarified. A previous study has revealed that p62 may play an important role in the antitumour activity of denosumab. The study aimed to investigate if the mechanism by which denosumab inhibits GCTB neoplastic stromal cells growth is via p62 modulation and other related mechanisms. p62 expression before and after denosumab therapy was analysed by RT‒qPCR, western blot, ELISA, and immunohistochemical assays. Two primary neoplastic stromal cells were isolated from fresh GCTB tumour tissue (L cell) and metastatic tissue (M cell). Cell proliferation, migration, apoptosis, and autophagy were investigated in p62 knockdown neoplastic stromal cells transfected by short hairpin RNA lentivirus in vitro. Tumor growth was evaluated in the chick chorioallantoic membrane model in vivo. p62 expression was found to be downregulated following denosumab therapy. The patients with a decrease in p62 expression had lower recurrence-free survival rates. The proliferation of M cells was not inhibited by denosumab therapy, but it was restored by p62 knockdown. Moreover, p62 knockdown inhibited tumour growth in vivo. Denosumab induced M cell apoptosis and arrested the cell cycle at the G1/G0 transition and these effects were also enhanced by p62 knockdown. Autophagic flux assays revealed p62 modulation to be dependent on autophagy following denosumab incubation. Denosumab induced neoplastic stromal cells apoptosis via p62 downregulation dependent on autophagy pathway. The combination of p62 and RANKL knockdown might be a better strategy than RANKL knockdown alone for GCTB targeted therapy.

MiRNA Signatures Related to Invasiveness and Recurrence in Patients With Non-Functioning Pituitary Neuroendocrine Tumors.

This preliminary study aimed to analyze and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary neuroendocrine tumors (NFPitNET). The relationship between deregulated miRNAs and tumor invasiveness, recurrence, and size was determined. Twenty patients with NFPitNET were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analyzed by quantitative real-time polymerase chain reaction (PCR) using LNA miRNA Cancer-Focus PCR Panel (Qiagen). Three miRNAs (miR-210-3p, miR-149-3p, and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPitNETs. Differential expression of four-miRNA signatures - miRNA-17, miR-19, miR-106a, and miR-20, correlated with patient recurrence. This prospective pilot study selected a unique miRNA expression profile, that correlates with invasiveness and recurrence in non-functioning pituitary neuroendocrine tumors. Moreover, some of the selected miRNAs are reported for the first time in patients with this disease, shedding light on the molecular mechanisms involved in pituitary pathogenesis. The identified miRNAs demonstrate potential as biomarkers, deserving further investigation in a larger cohort to validate their clinical applicability.

387 Pathogenicity of a CCDC6-RET Fusion in Malignant Peripheral Nerve Sheath Tumor (MPNST)

OBJECTIVES/GOALS: RETgene fusions in sarcoma are rare and their impact on pathogenicity is unknown. Malignant peripheral nerve sheath tumors (MPNST) are a deadly, genomically heterogenous soft tissue sarcoma rarely harboring targetable aberrations. We present a case of a CCDC6-RET fusion MPNST sensitive to RET-inhibitor therapy in a xenograft model. METHODS/STUDY POPULATION: Lung tumor tissue was obtained per an approved collection protocol from a 21yo male patient with a spontaneous MPNST harboring an inactivating mutation in NF-1 and a CCDC6-RET gene fusion detected by a commercially available sequencing panel (Signatera). To confirm pathogenicity of the RET fusion, fresh tumor tissue was engrafted into immunocompromised NSG mice in the anterior and posterior flanks, harvested at ~10 weeks, and re-transplanted into bilateral flanks. When tumor diameters reached 0.5-1cm (~4 weeks), mice were randomized into 3 groups (n=6/group) and treated with either vehicle (V) (PBS), the RET-specific inhibitor selpercatinib (S) (20mg/kg twice daily), or the multi-kinase inhibitor cabozantinib (C) (30mg/kg daily) by oral gavage. Mice were monitored weekly for weight and tumor size. RESULTS/ANTICIPATED RESULTS: 92% (33/36) of implanted tumors were evaluable for treatment response. Pre-treatment tumor volumes (mm3) across all three groups were similar (mean/Std Dev – V: 230/111, S: 271/132, C: 230/123). At day 7, tumor growth was significantly inhibited by S and C versus V (ANOVA p < 0.001, post-hoc Tukey’s V vs S p= 0.0178, V vs C p< 0.0001, S vs C p= 0.0005). V-treated tumors increased in volume by 60% while S reduced tumor volume by ~80% and C reduced tumor volume by ~20%. S and C treatments were tolerated well. and S improved survival with 100% of mice alive at day 63 vs 0% in V and C groups. 6 of the 12 implanted tumors treated with S, 50% increased in size after ~6-weeks following a >90% initial tumor reduction in tumor volume. Follow-on molecular studies in S-resistant tumors are ongoing. DISCUSSION/SIGNIFICANCE: Targetable genomic changes in MPNST, especially in RET, are infrequent and often considered stochastic. Our findings suggest that precision medicine approaches pairing genomic sequencing and in vivotesting of target gene pathogenicity may guide treatment planning and novel discovery for rare, difficult to treat sarcomas.

Abstract 4200: A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells

Abstract Human cancer tissue is a complex ecosystem with heterogeneous cell types that communicate with each other dynamically to promote tumor progression and immune evasion. Hence, experimental systems that destroy or select for cancer cells in culture have often failed to accurately predict clinical responses of new drugs. Therefore, a technology that can retain the intact tumor microenvironment (TME) and accurately predict cancer patient’s response to anti-cancer treatments, including immunotherapies, will greatly benefit patients and dramatically reduce the cost of new drug development. E-slice is a proprietary 3D human tumor culture platform that maintains individual patient tumor’s unique ecosystem and the TME ex vivo for up to 30 days. It is generated by making thin sections of intact, fresh tumor tissues and culturing them in serum-free, defined media. As such, immune components and the TME in E-slices faithfully recapitulate human tumors ex vivo. We demonstrate that E-slices can be used to: 1) measure viability changes upon anti-cancer agent treatment longitudinally; 2) retain the native TME and tissue architecture since E-slices are never dissociated or artificially reconstituted; 3) culture any solid tumor types from patient needle biopsy cores or surgical samples, PDX, or mouse models; 4) perform high content drug screening on human tissues; 5) discover secreted biomarkers and to 6) discover or validate molecular mechanisms of action or therapy resistance; and 7) measure immunotherapy responses ex vivo from human tissues. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-8 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. Furthermore, single cell RNA-sequencing analysis demonstrates that E-slices can retain the viability and molecular phenotypes of tumor infiltrating immune cells for up to 8 days ex vivo, providing a unique platform to study human tumor-immune interactions and evaluate efficacy of immune modulators on human tumor tissues ex vivo. Citation Format: Viridiana Leyva-Aranda, Thomas Gallup, Jose Maldonado, Corina Margain, Sang Yun, David Gallup, Min Kim, Kyuson Yun. A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells [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 4200.

Abstract 3748: Evaluation of immune cells from patient-derived tumor tissue and peritoneal washing cytology in correlation with immunohistochemical markers in endometrial cancer

Abstract Background This study aims to evaluate the characteristics and clinical correlations of tumor-infiltrating lymphocytes (TILs) in endometrial cancer, derived from fresh tumor tissue and peritoneal washing cytology. Furthermore, it seeks to explore the correlation between TILs and immunohistochemical markers in endometrial cancer. Methods In this study, we investigated the nature of TILs extracted from endometrial tissue and peritoneal washing cytology, along with their correlation with immunohistochemical markers. Samples collected from endometrial cancer patients during surgery, with informed consent, were analyzed for immune cell composition using flow cytometry. We examined the association between pathological findings, a range of immunohistochemical markers (including LVSI, PD-L1, ER, P53, HER2, MLH1, MSH2, MSH6, PMS2, MSI), and the status of tumor-infiltrating immune cells (such as CD3-CD19+ B Cells, CD3-CD16+CD56+ NK Cells, CD4+CD25+ regulatory T cells (Treg), and CD3+CD8+ cytotoxic T cells). Results Our analysis encompassed 25 endometrial cancer-derived samples. Notable observations include an increase in CD3-CD19+ B cells in the LVSI-positive group and a decrease in the ER-positive group (p<0.05, respectively). CD3-CD16+CD56+ NK Cells decreased in the ER-positive group, while CD3+CD8+ cytotoxic T cells were decreased in the HER2-positive group (p<0.001, p<0.05, respectively). Other immunohistochemical markers showed no significant differences. Conclusion The study suggests that tumor-infiltrating immune cells from patient-derived tissue and peritoneal washing cytology may reflect the molecular and clinical characteristics of endometrial cancer. However, further research with an expanded sample size and correlation with oncologic outcomes is warranted to validate these findings. Citation Format: Changmin Shin, Seungmee Lee, Hyewon Chung, Jin Young Kim, Eunyoung Ha, Ji Hae Seo, Sojin Shin. Evaluation of immune cells from patient-derived tumor tissue and peritoneal washing cytology in correlation with immunohistochemical markers in endometrial cancer [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 3748.

Abstract 6698: Tumor microbiota associated with immune enrichment in breast tumor

Abstract Asian cancer patients have enriched immune profiles when compared to Caucasians. This is associated with improved overall survival and potentially better response to immunotherapy. Yet the reason behind enriched Asian immune profiles is unclear. This study investigated microbes found in Malaysian breast tumors in association with immune profiles. We used an existing dataset of RNA-seq data from fresh frozen breast tumor tissue (n=978). Sequencing reads were trimmed and filtered to remove human reads. Non-human reads were mapped to Kraken2 and relative abundance was estimated using Bracken. Microbiome analysis was conducted on R following Microbiome OMA guidebook. The top five genera found in the Malaysian Breast Cancer (MyBrCa) cohort were Pseudomonas, Siphoviridae, Bacillus, Escherichia and Klebsiella. Alpha diversity of basal subtype microbiota was significantly higher than Her2 subtype (Shannon index, p=0.027) whilst the other subtypes were more homogenous. MyBrCa patients with low immune scores were significantly different from high immune scores while controlling for age, stage, grade, ethnicity and PAM50 subtype (PERMANOVA, F-statistic = 1.656, p=0.05). Differential abundance analysis revealed Sulfidibacter and Priestia to be significantly different in abundance between high vs low IFNg groups (FDR-adjusted p-value < 0.05) using ALDEx2, ANCOM-BC2 and ZicoSeq and LinDA algorithms. Sulfidibacter was significantly different between high vs low immune group for Bindea combined immune score and ESTIMATE score groups (FDR-adjusted p-value <0.05). An independent breast cancer cohort (n=419) was used as validation where Sulfidibacter and Priestia were also significantly different in abundance for immune high vs low groups. Machine learning using Random Forest model predicted immune group with an AUC of 0.79 (CI = 0.74, 0.83; p-value < 0.01). The top five most important features contributing to the random forest model were Sulfidibacter, Prestia, Cutibacterium, Acinetobacter and Rhizobium. This study is the largest to date that characterize the tumor microbiota of an Asian cohort of breast cancers, where Sulfidibacter and Priestia, both relatively new and uncharacterized bacteria, were differentially abundant among patients with high vs low immune scores. Citation Format: Li-Fang Yeo, Bernard KB Lim, Tania Islam, Li-Ying Teoh, Suniza Jamaris, Mee-Hoong See, Lai-Meng Looi, Nur Aishah Mohd Taib, Cheng-Har Yip, Pathmanathan Rajadurai, Soo-Hwang Teo, Jia Wern Pan. Tumor microbiota associated with immune enrichment in breast tumor [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 6698.