Paired Biopsy Samples Research Articles

From colon wall to tumor niche: Unraveling the microbiome's role in colorectal cancer progression.

Colorectal cancer (CRC) is influenced by perturbations in the colonic microbiota, characterized by an imbalance favoring pathogenic bacteria over beneficial ones. This dysbiosis contributes to CRC initiation and progression through mechanisms such as carcinogenic metabolite production, inflammation induction, DNA damage, and oncogenic signaling activation. Understanding the role of external factors in shaping the colonic microbiota is crucial for mitigating CRC progression. This study aims to elucidate the gut microbiome's role in CRC progression by analyzing paired tumor and mucosal tissue samples obtained from the colon walls of 17 patients. Through sequencing of the V3-V4 region of the 16S rRNA gene, we characterized the tumor microbiome and assessed its association with clinical variables. Our findings revealed a significant reduction in alpha diversity within tumor samples compared to paired colon biopsy samples, indicating a less diverse microbial environment within the tumor microenvironment. While both tissues exhibited dominance of similar bacterial phyla, their relative abundances varied, suggesting potential colon-specific effects. Fusobacteriota enrichment, notably in the right colon, may be linked to MLH1 deficiency. Taxonomy analysis identified diverse bacterial genera, with some primarily associated with the colon wall and others unique to this region. Conversely, several genera were exclusively expressed in tumor tissue. Functional biomarker analysis identified three key genes with differential abundance between tumor microenvironment and colon tissue, indicating distinct metabolic activities. Functional biomarker analysis revealed three key genes with differential abundance: K11076 (putrescine transport system) and K10535 (nitrification) were enriched in the tumor microenvironment, while K11329 (SasA-RpaAB circadian timing mediator) dominated colon tissue. Metabolic pathway analysis linked seven metabolic pathways to the microbiome. Collectively, these findings highlight significant gut microbiome alterations in CRC and strongly suggest that long-term dysbiosis profoundly impacts CRC progression.

Integrator complex subunit 6 promotes hepatocellular steatosis via β-catenin-PPARγ axis

Hepatic adipogenesis has common mechanisms with adipocyte differentiation such as PPARγ involvement and the induction of adipose tissue-specific molecules. A previous report demonstrated that integrator complex subunit 6 (INTS6) is required for adipocyte differentiation. This study aimed to investigate INTS6 expression and its role in hepatic steatosis progression. The expression of INTS6 and PPARγ was examined in the liver of a mouse model of steatohepatitis and in paired liver biopsy samples from 11 patients with severe obesity and histologically proven metabolic dysfunction associated steatohepatitis (MASH) before and one year after bariatric surgery. To induce hepatocellular steatosis in vitro, an immortalized human hepatocyte cell line Hc3716 was treated with free fatty acids. In the steatohepatitis mouse model, we observed hepatic induction of INTS6, PPARγ, and adipocyte-specific genes. In contrast, β-catenin which negatively regulates PPARγ was reduced. Biopsied human livers demonstrated a strong positive correlation (r2 = 0.8755) between INTS6 and PPARγ mRNA levels. After bariatric surgery, gene expressions of PPARγ, FABP4, and CD36 were mostly downregulated. In our in vitro experiments, we observed a concentration-dependent increase in Oil Red O staining in Hc3716 cells after treatment with the free fatty acids. Alongside this change, the expression of INTS6, PPARγ, and adipocyte-specific genes was induced. INTS6 knockdown using siRNA significantly suppressed cellular lipid accumulation together with induction of β-catenin and PPARγ downregulation. Collectively, INTS6 expression closely correlates with PPARγ. INTS6 suppression significantly reduced hepatocyte steatosis via β-catenin-PPARγ axis, indicating that INTS6 could be a novel therapeutic target for treating MASH.

Beacon: A phase II study of bevacizumab, atezolizumab, and cobimetinib in patients with recurrent, platinum resistant, high grade serous ovarian cancer.

5565 Background: Platinum resistant high grade serous ovarian cancer (HGSC) is associated with a poor prognosis and limited treatment options. Immune checkpoint inhibitors have failed to show significant impact in treatment of HGSC to date. Bevacizumab (anti-VEGF) and atezolizumab (anti-PDL1) have shown synergy in multiple cancer types. Cobimetinib (MEK inhibitor) may also potentiate immune responses, particularly by altering the tumour microenvironment. We sought to evaluate the efficacy of cobimetinib, bevacizumab and atezolizumab in women with platinum resistant HGSC. Methods: BEACON is a Phase II single arm study of cobimetinib (60mg oral D1-21 q 28-days), bevacizumab (5mg/kg IV q2w) and atezolizumab (840mg IV q2w from Cycle 2) in patients with platinum-resistant recurrent HGSC. Patients continued therapy until progression or unacceptable toxicity. The primary endpoint was overall response rate (ORR) according to RECIST 1.1 at 24 weeks. Secondary endpoints included best overall response (BOR), safety, progression free survival, and response duration. Correlative studies on paired tumor biopsy and blood samples will explore molecular characteristics/subtypes and potential biomarkers of response as exploratory objectives. This study has now completed accrual and we present the 24-week response data, with a data cut off 21st December 2023. Results: Twenty-nine patients were enrolled between September 2018 and June 2023. Median age was 62 years (range 37 - 79) and 97% had received ≥ 2 prior lines of chemotherapy. The median number of cycles received was 3 cycles of atezolizumab and cobimetinib, and 4 cycles of bevacizumab. The ORR at 24 weeks was 21% [95% CI: 8-40], and a further 28% had stable disease as assessed per RECIST. Five patients (17%) have remained on study treatment for ≥ 52 weeks to date, suggesting that in some patients there was durable disease control. In terms of safety, 22 patients (76%) required a dose reduction in cobimetinib, mainly due to gastrointestinal and skin toxicity. Seventeen patients (59%) experienced a ≥Grade 3 treatment related adverse event, most commonly hypertension (6 patients, 21%). Two patients (7%) discontinued all study treatments due to toxicity. Conclusions: Preliminary results from this study show promising efficacy with the combination of atezolizumab, bevacizumab and cobimetinib in platinum resistant HGSC. Duration of response, progression free survival data and translational data exploring molecular characteristics will be presented as data matures. Clinical trial information: NCT03363867 . [Table: see text]

Comparative genomic profiling of circulating tumor DNA and paired formalin-fixed paraffin-embedded (FFPE) DNA from Indian patients with breast cancer.

e14554 Background: Breast cancer (BC) is the most frequent malignancy and a leading cause of mortality in women worldwide. Although most BCs are treated with curative intent, 30-40 % patients experience disease progression or metastatic relapse. Considering high heterogeneity of BC, mutational profiling of solid biopsy may not be adequate for informed treatment decisions in all cases. Liquid biopsy based on circulating tumor DNA (ctDNA) analysis is emerging as an equivalent and alternative to tissue biopsy in clinical settings. Though extremely critical for management of BC, liquid biopsy is not a standard of care in India. Here we compared comprehensive genomic profiles of ctDNA and paired biopsy samples to determine the efficacy of ctDNA assay in detecting actionable mutational targets. Methods: Retrospectively, genomic DNA and matched ctDNA was isolated from both FFPE and blood samples of14 BC patients. The patients were confirmative with pathological findings, having metastatic infiltrating ductal carcinoma, or triple negative breast cancers. Genome-wide HRD, TMB and MSI scores based on the OncoIndx comprehensive gene panel (CGP) was determined. OncoIndx CGP targets 1080 cancer related genes along with 47 HRR genes and 1600 MSI signature sites. Libraries were prepared using a hybridization-capture method based on custom-designed OncoIndx 1080 CGP and sequenced (depth 5500 X) on Illumina Nextseq 2000 in a pair-end mode (150 x2). Variant calling was performed using a proprietary bioinformatics pipeline iCare. Results: All patients were diagnosed with metastatic infiltrating ductal carcinoma, or triple negative breast cancers. From the patient cohort, microsatellite instability (MSI) from ctDNA showed 100% concordance with FFPE DNA while tumor mutation burden (TMB) and homologous recombination deficiency (HRD) scores showed 71.4 % and 64.2 % concordance with FFPE DNA respectively. At the individual gene mutation level, the concordance TP53, BRCA1/2, PI3K family of genes, ESR1, PTEN, ERBB2, PALB2, and BRAF genes between ctDNA and FFPE DNA was 54.7%, 92.8%, 54.7%, 78.5%, 92.8%, 100%, 100%, and 100% respectively. Many important genomic alterations including PIK3CA:p.L334H, PIK3CA:p.E81K, ESR1:p.Q375L, BRCA2:p.X3040_splice, PTEN:p.D19H were exclusive to ctDNA thereby denoting potential tumor heterogeneities which could be missed from FFPE DNA. Conclusions: Our results suggest a high correlation between ctDNA and FFPE DNA detected by OncoIndx test in metastatic or triple negative breast cancer patients of Indian ethnicity. Although not a part of standard of care, liquid biopsy could be of significant potential for management of BC in the standard clinical settings. However, a thorough investigation of cancer stage, and clinical presentations are necessary to carefully evaluate the selection of ctDNA or FFPE DNA-based genomic profiling.

Abstract CT094: Targeting CSF1R with BLZ945 results in effective peripheral and tumor immune microenvironment modulation in advanced solid tumors and may be associated with limited efficacy in recurrent non-mesenchymal glioblastoma

Abstract Colony-stimulating factor 1 receptor (CSF-1R) signaling is involved in regulating monocyte differentiation, and the recruitment and survival of tumor-associated macrophages (TAMs). BLZ945 is a small molecule, highly selective, orally bioavailable, potent kinase inhibitor of CSF-1R that has shown the ability to reduce the recruitment of TAMs in animal models and promote infiltration of effector T cells into the tumor in preclinical models. Given the potential effects of BLZ945 on effector T-cell activity, a combination of BLZ945 with the programmed death 1 (PD-1) inhibitor spartalizumab was investigated in 1) a dose escalation phase I clinical trial with advanced solid tumor patients; and 2) an expansion phase II clinical trial in adult patients with relapsed/refractory glioblastoma (GBM) after standard of care and other lines of therapy (NCT02829723). The percentages of non-classical and intermediate monocytes in the PBMC fraction were decreased after treatment at multiple doses and schedules of treatment tested in the escalation phase. Screening and on-treatment paired tumor biopsy samples from the escalation phase were analyzed by RNA sequencing. Downregulated genes have been noted as macrophage-specific genes, suggesting that treatment with BLZ945 is downregulating macrophages in the tumor microenvironment. There appeared to be a trend for a reduction in TAM gene signatures and increase in T-cell inflammation signatures upon treatment with single-agent BLZ945 or BLZ945 + spartalizumab. RNA-sequencing analysis from archival surgical resection GBM samples from the expansion phase, revealed a potential correlation between PDGFRA gene expression and best percent change in tumor size. This suggests that non-mesenchymal GBM, for which PDGFRA is part of the defining signature, are more sensitive to the treatment. DNA-sequencing of these archival samples also revealed mutations in p53 in the tumors with partial responses but no mutations in EGFR or TERT. Citation Format: Carlos Rodrigo Gil Del Alcazar, Virginia Xu, Chia-chi Lin, Marta Gil-Martin, Aung Naing, Liqiong Fan, Fang Yang, David Quinn, Jincheng Wu, Cornelia Quadt, Jennifer Mataraza. Targeting CSF1R with BLZ945 results in effective peripheral and tumor immune microenvironment modulation in advanced solid tumors and may be associated with limited efficacy in recurrent non-mesenchymal glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT094.

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract 1765: The genetic landscape of head and neck cancer using brush biopsy

Abstract Oral premalignant lesions (OPLs) with genomic alterations have a heightened risk of evolving into oral squamous cell carcinoma (OSCC). Currently, genomic data are obtained through invasive tissue biopsy. Brush biopsy has been utilized for diagnosing dysplasia but its effectiveness in reflecting the complete genomic landscape of OPLs remains uncertain. This study investigates the potential of brush biopsy samples in accurately reconstructing the genomic profile of OPLs. The evolution and heterogeneity were assessed by assessing SNVs, copy number analysis, and subclonal architecture reconstruction of paired tissue-brush biopsy samples of oral epithelium, dysplastic lesion, and OSCC lesion. We found that brush biopsy accurately reflects the genomic landscape of oral lesions, mirroring about 90% of SNVs and comparable CNA profiles found in tissue biopsies. Genomic profiling with brush biopsy was tissue-specific, as SNVs identified in OPL or OSCC lesions were not found in adjacent normal mucosa. Shared SNVs and CNAs were observed between OPL and OSCC samples. This suggested a common ancestor giving rise to these lesions. Subclonal architecture reconstruction confirmed that both lesion types shared a common ancestor clone and then diverged evolutionarily. These findings underscore the potential of brush biopsies in accurately reconstructing the genomic profile of OPL and OSCC, highlighting their usefulness in understanding the biological processes involved in tumor evolution. Citation Format: Evit John, Tom Lesluyes, Toby Baker, Maxime Tarabichi, Peter Van Loo, Xiao Zhao. The genetic landscape of head and neck cancer using brush biopsy [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 1765.

CXCL10 and its receptor in patients with chronic hepatitis B and their ability to predict HBeAg seroconversion during antiviral treatment with TDF.

The serum chemokine C-X-C motif ligand-10 (CXCL10) and its unique receptor (CXCR3) may predict the prognosis of patients with chronic hepatitis B (CHB) treated with tenofovir disoproxil fumarate (TDF). Nevertheless, there are few reports on the profile of CXCL10 and CXCR3 and their clinical application in HBeAg (+) CHB patients during TDF antiviral therapy. CXCL10 and CXCR3 were determined in 118 CHB patients naively treated with TDF for at least 96 weeks at baseline and at treatment weeks 12 and 24. In addition, gene set enrichment analysis was used to examine the associated dataset from Gene Expression Omnibus and explore the gene sets associated with HBeAg seroconversion (SC). The change of CXCL10 (ΔCXCL10, baseline to 48-week TDF treatment) and CXCR3 (ΔCXCR3) is closely related to the possibility of HBeAg SC of CHB patients under TDF treatment. Immunohistochemical analysis of CXCL10/CXCR3 protein in liver tissue shows that there is a significant difference between paired liver biopsy samples taken before and after 96 weeks of successful TDF treatment of CHB patients (11 pairs) but no significance for unsuccessful TDF treatment (14 pairs). Multivariate Cox analysis suggests that the ΔCXCL10 is an independent predictive indicator of HBeAg SC, and the area under the receiver operating characteristic curve of the ΔCXCL10 in CHB patients is 0.8867 (p < 0.0001). Our results suggest that a lower descending CXCL10 level is associated with an increased probability of HBeAg SC of CHB patients during TDF therapy. Moreover, liver tissue CXCL10 might be involved in the immunological process of HBeAg SC.

Consistency and heterogeneity of microsatellite instability (MSI) status in paired biopsy and surgical specimens of colorectal cancer: A necessity for MSI reassessment after treatment?

149 Background: Microsatellite instability (MSI) plays a crucial role as a cancer immunotherapy and prognosis biomarker, making accurate MSI detection essential. However, currently, there is still a lack of available data on the impact of neoadjuvant therapy on MSI status, as well as validated data on standardized methods for MSI testing in small biopsy samples. The study aimed to investigate the concordance of MSI status between paired biopsy and surgical samples, as well as the impact of neoadjuvant therapy on MSI status using a novel MSI next-generation sequencing (MSI-NGS) detection panel. Methods: A total of 137 colorectal cancer (CRC) patients were enrolled for this study, of whom 116 with paired biopsy and surgical samples were analyzed. A custom MSI-NGS panel was employed and its performance was compared to MSI polymerase chain reaction (MSI-PCR), which served as the gold standard. Results: Out of the 116 cases, 112 patients showed consistent MSI status between biopsy and surgical samples, with an overall accuracy of 97% regardless of the detection method used. In the cases with MSI discrepancy (6 cases), 83% (5/6) of the patients showed a transition from MSS to MSI-H from biopsy to surgery. Interestingly, all eight patients who received neoadjuvant chemotherapy maintained an unchanged MSI status. However, in one patient who received adjuvant treatment and underwent a repeat biopsy after surgery, the MSI status exhibited alterations. Further analysis of clonal evolution revealed that this heterogeneity stemmed from the disappearance of the original clones and the emergence of new clones. Additionally, the NGS panel exhibited strong concordance with MSI-PCR and high accuracy, sensitivity and specificity with an AUC of 0.942 after rigorous validation. Conclusions: The study revealed a high concordance in MSI status between paired biopsy and surgical samples by employing a custom MSI-NGS panel for MSI status detection, providing reliable basis for further research in this field. Moreover, neoadjuvant chemotherapy seemed to have no impact on MSI status in our study, whereas postoperative adjuvant chemotherapy may influence changes in MSI status, highlighting the necessity of reevaluating MSI status after surgery. The findings also underscore the potential of NGS-based MSI detection as a valuable tool for clinical decision-making.

NNMT enriches for AQP5+ cancer stem cells to drive malignant progression in early gastric cardia adenocarcinoma

ObjectiveEarly gastric cardia adenocarcinoma (EGCA) is a highly heterogeneous cancer, and the understanding of its classification and malignant progression is limited. This study explored the cellular and molecular heterogeneity in...

Immunological Responses Associated With Neoadjuvant Therapy in the Tumor Microenvironment of Esophageal Squamous Cell Carcinoma.

Development of multidisciplinary therapies including immune checkpoint inhibitors for esophageal squamous cell carcinoma (ESCC) requires a clear understanding of immunological responses induced by chemotherapy with/without radiotherapy in the tumor microenvironment. This is a retrospective analysis of paired pretreatment biopsy samples and surgically resected tumor samples of 49 patients who underwent radical surgery for ESCC with/without neoadjuvant therapy at Fukushima Medical University Hospital. The cohort included 30 patients treated with neoadjuvant chemotherapy (NAC), 11 treated with neoadjuvant chemoradiotherapy (NACRT), and eight who underwent surgery alone and did not receive neoadjuvant antitumor therapy. Chemotherapy included fluoropyrimidine- and platinum-based agents in all treated patients, and radiotherapy included 40 or 42 Gy administered in 20 or 21 fractions. Expression of CD8, human leukocyte antigen (HLA) class I-ABC, PD-L1, PD-L2, CEACAM-1, LSECtin, and p-STAT1, were determined using immunohistochemistry. The frequency of tumor-infiltrating CD8+ T cells was significantly increased by NAC (p<0.05), and the expression of HLA class I-ABC on tumor cells was significantly increased by NAC and NACRT (p<0.05). Furthermore, the ESCC cells expressed PD-L1, PD-L2, and CEACAM-1, whereas the expression of PD-L1 on ESCC cells was significantly correlated with the expression of p-STAT1 in ESCC cells (p<0.05). NAC and NACRT induced both positive and negative immunological responses in patients with ESCC. These results may be a part of basis for multidisciplinary therapy including immune checkpoint inhibitors for patients with advanced ESCC.

Fibroblast inhibition by tocilizumab enabled gemcitabine/nab-paclitaxel rechallenge for pancreatic cancer.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC). We sought to determine the safety and recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with gemcitabine (GEM)/nanoparticle albumin-bound paclitaxel (nab-PTX)-refractory MPC. This phase 1 study enrolled 10 patients with MPC who had progressed after GEM/nab-PTX. Patients initially received TCZ 8 mg/kg on Day 1 and nab-PTX 100 mg/m2 + GEM 750 mg/m2 on Days 2, 9, and 16. Before and at the end of Cycle 1, biopsy of liver metastases was performed 3-5 h after levofloxacin (LVFX) administration to measure LVFX infiltration into tumor tissue. No dose-limited toxicities occurred, and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events occurred in 80% of patients, of which decreased neutrophil count was the most common. Tumor reduction during Cycle 1 was observed in four patients, who were defined as responders. In paired-biopsy samples, responder-related biological activities were an increase of cleaved PARP expression of tumor nuclei (p = 0.01), a decrease of proliferative cancer-associated fibroblasts (CAFs) (p = 0.08), and an increase of LVFX infiltration in the tumor (p = 0.04). A decrease of phosphorylated STAT3 expression (p = 0.02) favored an increase in LVFX infiltration. In conclusion, TCZ + GEM/nab-PTX-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and improved intratumoral drug infiltration in MPC.

Dynamic Profiling of the Immune Tumor Microenvironment in Locally Advanced Gastric Cancer Treated with Perioperative Chemotherapy

Introduction: In locally advanced gastric cancer (GC), FLOT represents the standard perioperative regimen and combination with immunotherapy is under investigation. However, the role of immune tumor microenvironment (TME) is poorly recognized in this setting. We aimed to study TME characteristics and dynamics during FLOT. Methods: Paired biopsy (PRE) and surgical (POST) samples of 25 patients treated with FLOT were prospectively analyzed. After collection of clinic-pathological data, NanoString analyses were performed. The primary objective of the study was to assess the changes induced by chemotherapy in POST compared to PRE samples. Results: The unsupervised hierarchical method analysis clearly distinguished PRE and POST samples, even though some cases showed high immune gene expression at baseline. When POST samples were compared with PRE, a differential expression in hyper-expressed gene sets related to cytotoxicity, T-cell functions, complement system, tumor necrosis factor superfamily, cell cycle, and regulation was recognized. Downstaging of the primary tumor (T-regression, measured by pathologic compared to clinical T stage) was the covariate most frequently associated with these changes. Using the immune cell profiling, cases with T-regression reported a significant increase of T, CD8+ T and B cells and a decrease in mast cells, while nonresponders demonstrated an increase of T, B, cytotoxic, and mast cells. Conclusion: Our analysis shows that FLOT significantly influences immune TME of GC. While relevant modifications preferentially occur in tumors showing primary tumor regression, response to treatment seems to be associated with a specific immune profile.

Lefitolimod (TLR agonist) and ipilimumab in patients with advanced solid tumors: A phase I trial.

2564 Background: The TLR-9 agonist lefitolimod (MGN1703) significantly increases the Th1 response in pre-clinical models and has demonstrated antitumor activity in early phase clinical trials. This trial assessed the safety and preliminary efficacy of the rational combination of lefitolimod with ipilimumab in patients (pts) with advanced solid tumors. Methods: This was a single-center, open-label, investigator-initiated, dose-finding, and expansion Phase I trial (ClinicalTrials.gov no. NCT0266877). Key inclusion criteria were histologically confirmed advanced solid tumor, ECOG performance status ≤ 2; age ≥ 18 years. Patients (pts) were enrolled at escalating doses of lefitolimod (15-120 mg/kg), subcutaneously (SC) administered weekly and a fixed dose of ipilimumab 3 mg/kg in a 3-week cycle. For the expansion cohort, lefitolimod was administered intratumorally (IT) at 15 mg weekly. The primary endpoint was safety and tolerability according to CTCAE v4.0, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD)/recommended Phase II dose (RP2D) for the lefitolimod in this combination. The secondary endpoint was efficacy, evaluated according to Immune-Related Response Criteria (irRC). Exploratory endpoints included changes in the intra-tumoral T-cell microenvironment in pre- and post- treatment tumor biopsies and the correlation of immune biomarkers with response. Results: From March 2017 to February 2020, 28 pts were enrolled. Median age was 56 years (range: 19-92). Median prior lines of therapy was 4 (range: 0-12). Pts 1-4 received lefitolimod 15 mg SC, pts 5-8 received lefitolimod 30 mg SC, pts 9-12 received lefitolimod 60 mg SC, pts 13-19 received lefitolimod 120 mg SC, and pts 20-28 received lefitolimod 15 mg IT. Eleven pts had at least one treatment-related AE (TRAE). The most common TRAE was skin rash (n=4, 14.2%), followed by fatigue (n=3, 10.7%) and pruritis (n=2, 7.1%). There were no grade 4 or 5 AEs, none of the pts required dose reduction, and there was no discontinuation of treatment due to AEs. DLT and MTD were not reached and the RP2D was established as lefitolimod SC 120 mg weekly with ipilimumab 3mg/Kg every 3 weeks. Of the 28 pts, 8 pts had stable disease as best response. 7 pts could not be evaluated by irRC due to clinical progression before restaging, non-measurable lesions and withdrawal of consent. Paired biopsy samples were analyzed by flow cytometry. Pro-inflammatory immune conditioning of the tumor microenvironment by the combination of the TLR9 agonist MGN1703 in combination with ipilimumab showed increases in intra-tumoral CD8 T cell frequency, memory CD8 phenotype (CD45RO+) and proliferation (Ki67+). Conclusions: The combination of high subcutaneous doses or intra-tumoral administration of lefitolimod in combination with ipilimumab is safe and well tolerated in patients with advanced cancers, with preliminary antitumor activity observed. Clinical trial information: NCT0266877 .

Ki67 increase after core needle biopsy associated with worse disease outcome in HER2-negative breast cancer patients

Ki67 would change after core needle biopsy (CNB) in invasive breast cancer. However, whether Ki67 alteration (ΔKi67) influences disease outcomes remains unclear. Here we aim to evaluate the prognostic value of ΔKi67. Patients with paired CNB and open excision biopsy (OEB) samples between January 2009 and June 2016 were retrospectively analyzed. ΔKi67 was calculated as the absolute difference between Ki67 level in CNB and OEB samples, and the median value of 5% was adopted to category patients into high- and low ΔKi67 groups. Disease-free survival (DFS) and overall survival (OS) were compared between different ΔKi67 groups. Overall, 2173 invasive breast cancer patients were included. Median Ki67 was higher in OEB than CNB samples: 25.00% versus 20.00% (P < 0.001). Axillary nodal status, STI, histological grading, and molecular subtype were independently associated with ΔKi67 (P < 0.05). In the whole population, patients with low ΔKi67 showed superior 5-year DFS (89.6% vs 87.0%, P = 0.026), but similar OS (95.8% vs 94.3%, P = 0.118) compared to those with high ΔKi67. HER2 status at surgery was the only significant factor interacting with ΔKi67 on both DFS (P = 0.026) and OS (P = 0.007). For patients with HER2-negative disease, high ΔKi67 was associated with worse 5-year DFS (87.2% vs 91.2%, P = 0.004) as well as impaired 5-year OS (93.9% vs 96.8%, P = 0.010). ΔKi67 had no significant impact on survival of HER2-positive patients. Ki67 increase after CNB was significantly associated with worse disease outcomes in HER2-negative, but not in HER2-positive patients, which warrants further study.

Impact of infiltrating T cell subtypes on survival and response to FLOT chemotherapy in resectable oesophagogastric adenocarcinoma (OGA).

443 Background: The prognostic and predictive relevance of tumour infiltrating T cell subtypes and of PD-L1 expression in localised OGA treated with FLOT chemotherapy (CTx) and surgery is poorly understood. Methods: Pre-CTx biopsy tissue was retrospectively collected from 46 patients (pts) with localised OGA who received perioperative FLOT and underwent surgery at a single institution. Opal Multicolour immunofluorescence was performed with primary antibodies against CD4, CD8, FOXP3, CD45RO, PD-1 and cytokeratin. Digital image analysis was performed using inForm and HALO softwares. PD-L1 CPS was determined using the E1L3N clone and the standard CPS assessment algorithm (CPS≥5 = high; CPS &lt;5 = low). CTx responses were assessed according to Mandard Tumour Regression Grade (TRG). For each immune cell marker and T cell phenotype, high vs low groups were defined using median cell density (count/mm2) as the cut-off. Association of high vs low cell density with disease free survival (DFS) was assessed using the log-rank test and correlation with pathological response to CTx was assessed using the Chi-squared test. The paired Wilcoxon rank-sum test was used to compare differences in immune cell densities between paired pre-CTx biopsy and post-CTx resection samples in a subset of 35 pts. Results: Pts with a PD-1+ cell density above median in biopsy had significantly longer DFS compared to those with a density below median (median DFS not reached vs 1111 days; HR 0.3, 95% CI 0.1 – 0.8; p=0.028). A trend towards longer survival was also observed in the CD8+ high, FOXP3+ high, and activated CD8+ (CD8+ PD-1+) high groups. There was no association between PD-L1 CPS, CD4+, CD45RO+, memory CD8+ (CD8+ CD45RO+), regulatory CD4+ (CD4+ FOXP3+), memory CD4+ (CD4+ FOXP3- CD45RO+), and activated CD4+ (CD4+ FOXP3- PD-1+) cells and survival. Pts with higher density of PD-1+ cells achieved better responses than those with low PD-1+ cells (TRG1/2 48% vs 9%, p=0.017). Pts with PD-L1 CPS ≥10 in biopsy were numerically more likely to achieve better responses to FLOT (46% TRG1/2). PD1+ cell density decreased in resection specimens in good CTx responders but did not differ in non-responders. FOXP3+ cells significantly decreased whereas CD45RO+ cells and memory CD8+ cells significantly increased in non-responders only. Conclusions: These results indicate prognostic and predictive value of PD-1 expressing tumour infiltrating T cells in resectable OGA treated with perioperative FLOT CTx. CD8+, activated CD8+ and FOXP3+ cells were associated with a trend in longer survival. This analysis provides candidate biomarkers for further study in larger cohorts on the association between infiltrating immune cells and prognosis as well as response to standard cytotoxic CTx in Western patients.

Uterine Cavity Lavage Mutation Analysis in Lithuanian Ovarian Cancer Patients.

Type II ovarian cancer (OC) is generally diagnosed at an advanced stage, translating into a poor survival rate. Current screening methods for OC have failed to demonstrate a reduction in mortality. The uterine lavage technique has been used to detect tumor-specific TP53 mutations from cells presumably shed from high-grade serous ovarian cancer (HGSOC). We aimed to pilot whether the detection of TP53 mutation in uterine cavity lavage can be used as a diagnostic method for HGSOC using an expanded gene panel. In this study 90, uterine lavage and 46 paired biopsy samples were analyzed using next-generation sequencing (NGS) targeting TP53 as well as five additional OC-related genes: BRCA1, BRCA2, PI3KCA, PTEN, and KRAS. Uterine lavage was successfully applied to all patients, and 56 mutations were detected overall. TP53 mutations were detected in 27% (10/37) of cases of type HGSOC; BRCA1 and BRCA2 mutations were also frequent in this group (46%; 17/37). Overall concordance between tissue and liquid biopsy samples was 65.2%. Uterine lavage TP53 mutations in combination with other biomarkers could be a useful tool for the detection of lowly invasive HGSOC.

Chronic Kidney Disease Is Characterized by Expansion of a Distinct Proinflammatory Intermediate Monocyte Subtype and by Increased Monocyte Adhesion to Endothelial Cells.

CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 β than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.

Biomarker Analysis of Zanubrutinib and Tislelizumab Combination Therapy in Patients with Relapsed/Refractory B-Cell Malignancies

Background/Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma world-wide. Although frontline therapies have yielded positive outcomes in most patients with DLBCL, about one-third of patients are refractory to or relapse after standard therapy (Sehn et al. J Clin Oncol 2005;23(22):5027-33). The antitumor efficacy of zanubrutinib and tislelizumab combination therapy has been demonstrated in patients with B-cell malignancies (Tam et al. Blood 2019;134:1594). Herein, we performed a comprehensive analysis to examine biomarkers associated with response or resistance to zanubrutinib and tislelizumab combination therapy in patients with B-cell malignancies. Methods: Biomarker analysis was performed in 24 patients with published response data determined by the investigator using Lugano 2014 criteria (Cheson et al. J Clin Oncol 2014;32(27):3059-68). Programmed death-ligand 1 (PD-L1) gene amplification was assessed in the baseline tumor of 11 patients with non-germinal center B-cell (GCB) subtype of DLBCL by fluorescence in situ hybridization (Empire Genomics). PD-L1 protein expression was examined in the baseline tumor (8 DLBCL) by immunohistochemistry (IHC) (Ventana PD-L1 [SP263] assay; Roche). DLBCL subtype and gene expression were examined in the baseline tumor (14 DLBCL) by HTG EdgeSeq DLBCL cell of origin assay (HTG Molecular Diagnostics). Gene mutation was examined in the baseline tumor (17 DLBCL) by DNA-seq (CGI NGS 220-Gene Panel assay; Cancer Genetic Inc.). PD-L1 and CD8 protein expression were examined in paired biopsy samples (1 DLBCL, 3 transformed follicular lymphoma [tFL], 1 follicular lymphoma [FL], 1 mantle cell lymphoma) before and 8 days after zanubrutinib treatment by IHC (Ventana SP263, CONFIRM CD8 SP57). Gene expression profiles were examined in paired biopsy samples (3 tFL, 1 FL, 1 chronic lymphocytic leukemia) by RNA-seq (RNA Access assay; Illumina). Results:PD-L1 gene alteration was observed in 2 of 11 patients with non-GCB DLBCL, including 1 with gene amplification and 1 with chromosome 9 polysomy. A higher overall response rate (ORR; 2/2 [100%] vs 3/9 [33.3%]) and complete response (CR) rate (2/2 [100%] vs 2/9 [22.2%]) was observed in patients with PD-L1 gene alteration than in patients without. Two of 8 [25%] evaluable patients with DLBCL harbored PD-L1+ tumor cells (defined using a PD-L1 protein expression cut-off ≥1%). A higher ORR (1/2 [50%] vs 2/6 [33%]) and CR rate (1/2 [50%] vs 1/6 [16.7%]) was observed in patients with PD-L1+ tumor cells than in those without. High mRNA levels of CD3D (P < 0.05), HLA-DRA (P < 0.07), and LAG3 (P < 0.05) were enriched in responders, suggesting an inflamed tumor microenvironment (TME). In contrast, non-responders harbored high mRNA levels of the NF-kB pathway-related gene REL (P < 0.05). High mRNA levels of REL were associated with an inferior clinical outcome to zanubrutinib and tislelizumab combination therapy (P < 0.05). Mutations in the tumor suppressor gene TP53 (5/10 [50%] vs 0/7 [0%]) were enriched in non-responders. Non-responders harbored higher frequency of mutations in genesinvolved in immune evasion (3/10 [30%] vs 1/7 [14.3%]), epigenetic modifications (2/10 [20%] vs 1/7 [14.3%]), and cell survival (2/10 [20%] vs 0/7 [0%]) than responders, suggesting that non-responders may have complex resistance mechanisms. Results from paired tumor biopsies from 5 patients before and after zanubrutinib treatment revealed that zanubrutinib treatment downregulated B-cell and MYC target-related gene sets and upregulated the NK cell-related gene set NK-2. Zanubrutinib treatment seemed to have no effect on the frequency of PD-L1+ cells and CD8+ T cells (using a CD8 expression cutoff of ≥1%) in the TME, although the sample size was too small to draw a definitive conclusion. Conclusion: Patients with PD-L1 gene amplification, PD-L1+ tumor cells, and high mRNA levels of CD3D, HLA-DRA, and LAG3 in baseline tumor tissue may be more responsive to zanubrutinib and tislelizumab combination therapy. A high mRNA level of REL or mutations in TP53 may contribute to resistance of zanubrutinib and tislelizumab combination therapy. Due to the limited number of samples, results must be interpreted with caution.

CD8 Presence and Dynamics in Glofitamab-Treated Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma Using the CD8-Specific PET Tracer 89Zr-Crefmirlimab Berdoxam

Background: Glofitamab, a CD20xCD3 T-cell-engaging bispecific monoclonal antibody with a novel 2:1 (CD20:CD3) configuration, redirects T cells to eliminate normal and malignant B cells. In ongoing Phase I/II trials, glofitamab has shown high response rates and durable complete responses in patients with relapsed/refractory (R/R) B-cell lymphomas (Hutchings et al. 2021, J Clin Oncol), including diffuse large B-cell lymphoma (DLBCL; Dickinson et al. ASCO 2022). In this imaging study (NCT03533283), the glofitamab-mediated effects on the distribution of CD8-expressing cells, such as cytotoxic T cells, in patients with R/R B-cell non-Hodgkin lymphoma (NHL) were captured with the novel 89Zr-radiolabeled anti-CD8 humanized minibody crefmirlimab berdoxam (CD8 tracer) for positron emission tomography (PET). Methods: Patients received obinutuzumab (1000 mg) once 7 days before the 1st glofitamab dose; glofitamab was then given as step-up doses on Day 1 (2.5 mg) and Day 8 (10 mg). The baseline infusion of the CD8 tracer was administered prior to obinutuzumab and the on-treatment infusion of the CD8 tracer was performed 48 h (±24 h) after the 1st or 2nd glofitamab dose. The tracer was infused at a fixed dose of 37 MBq (1.5 mg protein); PET/CT scans were performed 24 h post infusion. The primary endpoint was quantification of changes of CD8 tracer uptake (SUVmax) in tumor lesions before and after glofitamab treatment. Paired biopsies for CD8 immunohistochemistry (IHC) and serum pharmacokinetic samples were taken at baseline and on-treatment. Results: To date, 9 evaluable patients with a median of 2 prior treatment lines (range: 1-6) with B-cell NHL (follicular lymphoma (FL), n=5; transformed FL, n=1; DLBCL, n=3) were enrolled. CD8 tracer infusions were well tolerated with no reported tracer-related adverse events. No difference in serum clearance of the CD8 tracer was observed between baseline and on-treatment infusions. Tracer uptake was detected in CD8 rich tissues (e.g. spleen, bone marrow, lymph nodes) and clearance organs (liver, kidney, intestine). Three patients with DLBCL received on-treatment CD8-PET/CT scans after the 2nd glofitamab dose. 11 analyzed DLBCL lesions showed variable CD8 tracer uptake at baseline between SUVmax 2.6-38.27. Following treatment with glofitamab, the signal in 64% of the analyzed lesions was increased 1.2-3.3-fold while the signal in 18% of the lesions was stable and 18% showed a decreased signal compared to baseline. The distribution of the CD8 tracer in individual lesions was highly heterogeneous with areas of low and higher uptake (Fig. 1A). The CD8 IHC of paired biopsy samples taken after PET scans in 1 patient with DLBCL showed good correspondence between PET and IHC signal. The on-treatment biopsy contained quantifiable T cell amounts without detection of viable tumor tissue, suggesting a fast onset of glofitamab activity prior to the selected imaging/biopsy timepoint. 6 patients with FL were analyzed. In 87% of the analyzed 39 FL lesions, a high and heterogenous CD8-tracer uptake between SUVmax 5.3-29.2 was observed at baseline. After one dose of 2.5 mg glofitamab, 36% of the analyzed lesions showed a stable or ≤1.3-fold increase of CD8-tracer uptake. In 64% of the analyzed lesions a ≤2.1-fold decrease of CD8-tracer uptake was observed (Fig.1B). After the 2nd glofitamab dose, CD8-PET-scans of patients with FL revealed that in 86% of the analyzed lesions CD8 tracer uptake decreased 1.2-11.9-fold compared with baseline scans, accompanied by shrinkage of lesion size as detected on low dose CT. Conclusions: Changes in the uptake of the CD8 tracer were observed early after treatment with glofitamab. Preliminary results suggest indication-specific differences in T-cell dynamics after two treatment interventions with glofitamab. In DLBCL, the CD8 tracer confirmed recruitment of T cells to the majority of tumor lesions after two glofitamab treatments. In FL, the preliminary CD8-tracer results suggest that the intra tumoral T cells exhibit their antitumoral effects immediately after glofitamab administration and thus faster compared to the glofitamab-mediated effects in DLBCL. In addition these data indicate that the optimal imaging time point is after the 1st glofitamab dose. 89Zr-crefmirlimab berdoxam is a promising innovative tool to assess the presence and dynamic change of CD8 T cells after treatment with novel immunotherapies for hematological malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal