Tumor Sections Research Articles

Correlation of tumoral collagen width with overall survival and tumoral collagen straightness with distant recurrence-free survival in patients with pancreatic ductal adenocarcinoma.

779 Background: Quantitative measurements of fibrillar collagen characteristics has been shown to correlate with survival in several types of malignancy. The aim of this study was to determine whether these characteristics correlate with survival in pancreatic ductal adenocarcinoma. Methods: Patients who underwent pancreatic resection for pancreatic ductal adenocarcinoma at an Allegheny Health Network hospital between 1/1/2019 and 12/31/2023, and for whom archived formalin-fixed paraffin-embedded tumor tissue was available, were included in the study. Clinical and pathologic characteristics were collected from the patient's electronic medical record. Survival data included overall survival (OS), local recurrence-free survival (RFS), and distant RFS. One section of tumor per patient was stained with Picrosirius Red and a representative digital image was obtained with a camera attached to a microscope at 100x magnification. Each image was analyzed with CT-FIRE software to determine averages of collagen fiber width, length, straightness, and angle. Descriptive statistics were initially computed. Log-rank tests were also conducted to compare the OS rate and RFS rate between four different groups (width, length, straightness, angle). Each group was divided into high and low groups, using the mean as cutoff. In the end, multivariate Cox regression was conducted by controlling the confounders including sex, perineural invasion, lymphovascular invasion, and presence of lymph node metastasis to examine the hazard ratios of OS, local RFS, and distant RFS for the four collagen fiber metrics. All statistical analyses were performed via SAS 9.4 under alpha level .05. Results: One hundred three patients met the inclusion criteria. Based on the results of log-rank test, OS was significantly different between high and low collagen width groups, p = .0251. The lower width group had higher survival rate than the upper width group. Also, distant recurrence-free survival was significantly different between high and low collagen straightness groups, p = .0053. The lower straightness group had higher distant RFS rate than upper group. The results of multivariate Cox regression were consistent with the Kaplan-Meier curves. Compared to the high collagen width group, the low collagen width group had a 57% lower chance of dying, hazard ratio = 0.43, 95% CI [0.23, 0.78], p = .0056. The low collagen straightness group had a 60% lower chance of distant recurrence than the high collagen straightness group, hazard ratio = 0.40, 95% CI [0.19, 0.85], p =.0163. Conclusions: Quantitative collagen fiber metrics could help identify patients with resected pancreatic adenocarcinoma who are at risk of earlier death and distant recurrence.

A multimodal deep learning approach for the prognostic stratification of patients with colorectal cancer (CRC).

287 Background: Traditional prognostic systems like the AJCC TNM staging for colorectal cancer (CRC) often fall short in predicting long-term patient outcomes. These systems often rely on limited pathologic features, leading to generalized approaches to treatment despite diverse tumor heterogeneity, such as the complex interaction between the tumor-host microenvironment. There is therefore a pressing need for more accurate, scalable tools to enhance decision-making and predict outcomes for patients with CRC. Methods: Tumor sections from 191 CRC patients were comprehensively stained for CD8 to examine CD8-positive tumor-infiltrating lymphocytes (TILs). To account for tumor heterogeneity, multiple areas of the tumor were evaluated to include intertumoral and peritumoral areas. Slides were digitized and quantitatively analyzed using a cloud-based platform (SpatialX Diagnostics, Inc. USA). Additionally, clinicopathologic features including patient demographics, 8th edition AJCC staging, lymphovascular invasion (LVI), perineural invasion (PNI), mismatch repair (MMR) protein status, tumor location, presence/development of distant metastases, and patient follow-up were collected. Merging CD8 AI findings with clinicopathologic features, a complete data set was available for 175 patients. Cases were randomly assigned into 70% training and 30% validation. In the training set, unsupervised artificial intelligence (AI) models integrated clinicopathologic data and CD8-positive TIL counts using vision transformer models. The final model combined spatially resolved pathologic features with clinical variables to statistically predict overall survival (OS) and distant metastasis risk (DMR). Results: In the validation cohort, the model stratified patients into two groups based on a deep learning-derived risk score, with statistically significant differences between low-risk and high-risk groups for OS (p<0.02) and DMR prediction (p<0.0001), For OS prediction, the model selected tumor site, PNI, MMR status, epithelial and stromal CD8-positive TILs, and 5 deep vision features as the top 10 clinically significant features. For DMR prediction, the age, T- and N-stage, PNI, and 6 deep vision-derived features as key predictors. Conclusions: As captured by vision transformers, the distribution and spatial arrangement of CD8-positive TILs are critical factors in patient stratification for OS and DMR. AI-driven models like the one herein offer the potential for more personalized management strategies, advancing CRC management and improving patient outcomes beyond traditional prognostic staging systems and parameters.

The prevalence of Lynch syndrome and associated clinicopathologic features in patients with mismatch repair deficient gastric cancer.

380 Background: Approximately 2-4% of patients with primary colorectal cancer are estimated to be associated with Lynch syndrome. However, the prevalence of Lynch syndrome and associated clinicopathologic features in patients with mismatch repair-deficient (dMMR) gastric cancer (GC) is not well understood. In this study, we investigated the frequency of dMMR GCs in unselected GCs and the prevalence of Lynch syndrome, as well as associated clinicopathologic features and molecular alterations in dMMR GCs. Methods: A total of 847 cases of unselected GCs from 2010 to 2012 were collected at Jeju National University Hospital. Immunohistochemistry for mismatch repair proteins (MMR), including MLH1, MSH2, MSH6, and PMS2, and MSI analysis were performed on representative tumor sections of surgically resected specimens. Follow-up genetic counseling and genetic and/or epigenetic alterations of the MMR genes were also investigated. Results: Loss of one or more MMR proteins and microsatellite instability-high were observed in 72 patients; 67 patients showed loss of MLH1/PMS2, 2 patients showed loss of PMS2 alone, and 3 patients with MSH2/MSH6. Genetic counseling demonstrated that ten patients had a family history of LS-associated cancers and nine patients had a history of metasynchronous cancers. Of the 72 patients, 65 revealed hypermethylation of the promoter region of MLH1. The subsequent genetic germline tests demonstrated 2 patients with MLH1 mutation, 3 with MSH2 mutation, and 2 with PMS2 mutation. Conclusions: dMMR tumors were identified in 72 (8.5%) of 847 unselected primary GCs. This study estimated that 0.8% of patients with unselected GC and 9.7% of patients with dMMR GC fulfilled the revised Bethesda criteria and are associated with Lynch syndrome. The detection of dMMR GCs and necessary genetic counseling/tests based on revised Bethesda criteria would help find Lynch syndrome in patients with GC. Clinical and immunophenotypical features of patients with dMMR GC. Age ≤ 50 years 5 (6.9) > 50 years 67 (93.1) Other malignancy history except GAC No 63 (87.5) Yes 9 (12.5) History of metachronous cancers Esophageal cancer 1 (1.4) Maxillary sinus cancer 1 (1.4) Papillary thyroid cancer 1 (1.4) Colorectal cancer 1 (1.4) Pancreatic cancer 1 (1.4) Breast cancer 1 (1.4) Breast cancer and malignant GIST 1 (1.4) Multiple HCCs 2 (2.8) History of Family with LS related cancers No 62 (86.1) Yes 10 (13.9) Fulfillment of the rBG No 63 (87.5) Yes 9 (12.5) MMR proteins Loss of MLH1 and PMS2 67 (93.1) Loss of PMS2 2 (2.8) Loss of MSH2 and MSH6 3 (4.2) Germline mutation test MLH1 mutations 2 (2.8) MSH2 mutations 3 (4.2) PMS2 mutations 2 (2.8)

DNA-Dependent Protein Kinase Inhibitor Peposertib Enhances Efficacy of 177Lu-Based Radioimmunotherapy in Preclinical Models of Prostate and Renal Cell Carcinoma.

Novel radiation sensitizers, including inhibitors targeting DNA damage response, have been developed to enhance the efficacy of anticancer treatments that induce DNA damage in cancer cells. Peposertib, a potent, selective, and orally administered inhibitor of DNA-dependent protein kinase, impedes the nonhomologous end-joining mechanism for DNA double-strand break (DSB) repair. We investigated radioimmunotherapy alone or with peposertib in preclinical models of renal cell carcinoma (RCC) or prostate cancer. Methods: 177Lu-DOTA-girentuximab (targeting carbonic anhydrase IX) or 177Lu-DOTA-rosopatamab (targeting prostate-specific membrane antigen) was used to deliver β-radiation to tumors via a single intravenous dose (3 or 6 MBq) in mice bearing SK-RC-52 RCC or LNCaP prostate cancer xenografts, respectively. Peposertib (50 mg/kg daily for 14 d) was administered via oral gavage. Biodistribution and invivo imaging of 177Lu-based radioimmunotherapy were performed for both preclinical models. Tumor growth and body weight were monitored until the endpoint. Assessment of DNA damage was performed by measuring DSBs through analysis of γH2AX foci formation in tumor sections. Results: Ex vivo biodistribution and invivo SPECT/MRI revealed excellent tumor uptake of each radiopharmaceutical. Mouse body weight was stable in all treatment arms. Peposertib alone did not show a significant antitumor effect. The addition of peposertib to 177Lu-DOTA-girentuximab showed enhanced antitumor efficacy compared with 177Lu-DOTA-girentuximab alone in the SK-RC-52 animal model, with a 4 of 4 complete response rate in the 177Lu-DOTA-girentuximab (6 MBq) plus peposertib arm. Peposertib combined with low-dose 177Lu-DOTA-girentuximab (3 MBq) demonstrated antitumor activity comparable to 177Lu-DOTA-girentuximab (6 MBq) monotherapy. In the LNCaP prostate cancer model, the combination of 177Lu-DOTA-rosopatamab (6 MBq) and peposertib achieved a 3 of 4 complete response rate. Increased DSBs were observed with the addition of peposertib to 177Lu-based radioimmunotherapy. Conclusion: The combination of peposertib with 177Lu-based radioimmunotherapy was well tolerated in preclinical models of RCC and prostate cancer. Our findings suggest a synergistic effect between peposertib and 177Lu-based radioimmunotherapy, wherein peposertib enhanced the efficacy of radioimmunotherapy. This synergy indicates the potential to reduce the necessary dose of radioimmunotherapy for effective cancer treatment.

Spatial transcriptome reveals histology-correlated immune signature learnt by deep learning attention mechanism on H&E-stained images for ovarian cancer prognosis

BackgroundThe ability to predict the prognosis of patients with ovarian cancer can greatly improve disease management. However, the knowledge on the mechanism of the prediction is limited. We sought to deconvolute the attention feature learnt by a deep learning convolutional neural networks trained with whole-slide images (WSIs) of hematoxylin-and-eosin (H&E)–stained tumor samples using spatial transcriptomic data.MethodsIn this study, 773 WSIs of H&E-stained tumor sections from 335 patients with treatment naïve high-grade serous ovarian cancer who were included in The Cancer Genome Atlas (TCGA) Pan-Cancer study were used to train, and validate, and to test a ResNet101 CNN model modified with attention mechanism. WSIs from patients in an independent cohort were used to further evaluate the model.ResultsThe prognostic value of the predicted H&E-based survival scores from the trained model on patient survival was evaluated. The attention signals learnt by the model were then examined their correlation with immune signatures using spatial transcriptome. After validating the model with the testing datasets, pathway enrichment analysis showed that the H&E—based survival score significantly correlated with certain immune signatures and this was validated spatially using spatial transcriptome data generated from ovarian cancer FFPE samples by correlating the selected signature and attention signal.ConclusionsIn conclusion, attention mechanism might be useful to identify regions for their specific immune activities. This could guide future pathological study for the useful immunological features that are important in modulating the prognosis of ovarian cancer patients.

Unveiling Microscopic Variations during Photodynamic Therapy via Polarity-Responsive Fluorescence Lifetime Imaging.

Photodynamic therapy is a highly promising method for cancer adjuvant treatment. However, the current research on the microscopic changes during the photodynamic therapy process is still quite limited, which seriously impedes the deep understanding of the procedure. For this purpose, a novel polarity-responsive probe, MI-PPF, with excellent mitochondrial targeting and anchoring capabilities has been rationally designed and synthesized. Notably, MI-PPF has successfully realized the in situ detection of mitochondrial morphology and polarity alterations during the photodynamic therapy process in cancer cells through fluorescence lifetime imaging. The results showed that a series of phenomena such as deformation, shrinkage, vacuolation, and aggregation occurred in the mitochondrial morphology during photodynamic therapy. Concurrently, a decline in mitochondrial polarity is also noted, which may be closely linked to the mitochondrial oxidative stress response during this process. Furthermore, MI-PPF can be used for photodynamic therapy on tumor mouse models and has successfully achieved fluorescence lifetime imaging of tumor sections before and after photodynamic therapy, uncovering multifaceted changes in cell morphology, polarity, and polarity distribution within the mouse tumor model during the process. It is anticipated that this study will offer valuable insights and guidance to the research of mitochondrial-related fields and will boost the advancement of diagnostic and therapeutic areas for associated diseases.

A subtype specific probe for targeted magnetic resonance imaging of M2 tumor-associated macrophages in brain tumors.

Pro-tumoral M2 tumor-associated macrophages (TAMs) play a critical role in the tumor immune microenvironment (TIME), making them an important therapeutic target for cancer treatment. Approaches for imaging and monitoring M2 TAMs, as well as tracking their changes in response to tumor progression or treatment are highly sought-after but remain underdeveloped. Here, we report an M2-targeted magnetic resonance imaging (MRI) probe based on sub-5 nm ultrafine iron oxide nanoparticles (uIONP), featuring an anti-biofouling coating to prevent non-specific macrophage uptake and an M2-specific peptide ligand (M2pep) for active targeting of M2 TAMs. The targeting specificity of M2pep-uIONP was validated in vitro, using M0, M1, and M2 macrophages, and in vivo, using an orthotopic patient-tissue-derived xenograft (PDX) mouse model of glioblastoma (GBM). MRI of the mice revealed hypointense contrast in T2-weighted images of intracranial tumors 24 h after receiving intravenous (i.v.) injection of M2pep-uIONP. In contrast, no noticeable contrast change was observed in mice receiving scrambled-sequence M2pep-conjugated uIONP (scM2pep-uIONP) or the commercially available iron oxide nanoparticle formulation, Ferumoxytol. Measurement of nanoparticle-induced T2 value changes in tumors showed 38 %, 9 %, and 2 % decrease for M2pep-uIONP, scM2pep-uIONP, and Ferumoxytol, respectively. Moreover, M2pep-uIONP exhibited 88.7-fold higher intra-tumoral accumulation compared to co-injected Ferumoxytol at 24 h post-injection. Immunofluorescence-stained tumor sections showed that CD68+/CD163+ M2 TAMs were highly co-localized with Cy7-M2pep-uIONP, but not with Cy7-scM2pep-uIONP and Cy7-Ferumoxytol. Flow cytometry analysis revealed 26 ± 10 % of M2 TAMs were targeted by M2pep-uIONP, which was significantly higher than Ferumoxytol (16 ± 1 %) and scM2pep-uIONP (13 ± 4 %) with the same dosage (20 mg Fe/kg). These findings demonstrate that M2pep-uIONP functions as a ligand-mediated MRI probe for targeted imaging of M2 TAMs in GBM, with potential applications for imaging of M2 TAM in other cancer types. STATEMENT OF SIGNIFICANCE: Targeting the pro-tumoral M2 subtype of tumor-associated macrophages (TAMs) to modulate the tumor immune microenvironment (TIME) is an emerging strategy for developing novel cancer therapies and enhancing the efficacy of existing treatments. In this study, we have developed a magnetic resonance imaging (MRI) probe using sub-5 nm ultrafine iron oxide nanoparticles (uIONP), which are coated with an anti-biofouling polymer and conjugated to an M2-specific peptide ligand (M2pep). Our results demonstrate that M2pep-uIONP exhibits an 88.7-fold higher accumulation in intracranial tumors in an orthotopic patient-derived xenograft (PDX) model of glioblastoma compared to the commercial iron oxide nanoparticle, Ferumoxytol. This enhanced accumulation enables M2pep-uIONP to induce significant MRI contrast, providing a non-invasive imaging tool to visualize M2 TAMs and monitor changes in the TIME of brain tumors and potentially other cancers.

Oral delivery of MOMIPP lipid nanoparticles for methuosis-induced cancer chemotherapy.

Methuosis, a non-apoptotic pattern of cell death, triggers the accumulation of macropinosome-derived vacuoles in the cytoplasm. Through this novel mechanism, methuosis inducers possess great potential in fighting apoptosis-resistant cancer cells and offer a promising alternative for cancer treatment. However, the potent methuosis inducer, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), faces an intractable issue of insolubility in most solvents, hindering in vivo dosing and compromising the validation of its antitumor efficacy. Few strategies have been developed to effectively deliver MOMIPP and achieve robust in vivo tumor inhibition since its first report in 2012. Here, a MOMIPP self-emulsifying drug delivery system (MOMIPP-SEDDS) was developed to substantially improve its oral bioavailability and achieve a favorable antitumor effect in a mouse xenograft tumor model. Our findings demonstrated that the MOMIPP-SEDDS was internalized into Caco-2 cells via the lipid raft/caveolae pathway and exhibited enhanced absorption in both cell monolayers and everted gut sacs. Compared with MOMIPP suspensions, MOMIPP-SEDDS showed a 13.3-fold increase in peak concentration and increased relative bioavailability by 19.98 times. By inducing methuosis, MOMIPP-SEDDS successfully retarded tumor progression in a subcutaneous HeLa mouse tumor model. Additionally, transmission electron microscopy (TEM) images of the tumor sections evidenced the occurrence of methuosis in the MOMIPP-SEDDS treatment group. This MOMIPP-SEDDS emerges as a promising lipid nanoparticle platform and high translational medicine for the oral delivery of MOMIPP to exert methuosis-induced tumor suppression for cancer treatment.

Deciphering a profiling based on multiple post-translational modifications functionally associated regulatory patterns and therapeutic opportunities in human hepatocellular carcinoma

BackgroundPosttranslational modifications (PTMs) play critical roles in hepatocellular carcinoma (HCC). However, the locations of PTM-modified sites across protein secondary structures and regulatory patterns in HCC remain largely uncharacterized.MethodsTotal proteome and nine PTMs (phosphorylation, acetylation, crotonylation, ubiquitination, lactylation, N-glycosylation, succinylation, malonylation, and β-hydroxybutyrylation) in tumor sections and paired normal adjacent tissues derived from 18 HCC patients were systematically profiled by 4D-Label free proteomics analysis combined with PTM-based peptide enrichment.ResultsWe detected robust preferences in locations of intrinsically disordered protein regions (IDRs) with phosphorylated sites and other site biases to locate in folded regions. Integrative analyses revealed that phosphorylated and multiple acylated-modified sites are enriched in proteins containing RRM1 domain, and RNA splicing is the key feature of this subset of proteins, as indicated by phosphorylation and acylation of splicing factor NCL at multiple residues. We confirmed that NCL-S67, K398, and K646 cooperate to regulate RNA processing.ConclusionTogether, this proteome profiling represents a comprehensive study detailing regulatory patterns based on multiple PTMs of HCC.

AxonFinder: Automated segmentation of tumor innervating neuronal fibers.

Neurosignaling is increasingly recognized as a critical factor in cancer progression, where neuronal innervation of primary tumors contributes to the disease's advancement. This study focuses on segmenting individual axons within the prostate tumor microenvironment, which have been challenging to detect and analyze due to their irregular morphologies. We present a novel deep learning-based approach for the automated segmentation of axons, AxonFinder, leveraging a U-Net model with a ResNet-101 encoder, based on a multiplexed imaging approach. Utilizing a dataset of whole-slide images from low-, intermediate-, and high-risk prostate cancer patients, we manually annotated axons to train our model, achieving significant accuracy in detecting axonal structures that were previously hard to segment. Our method achieves high performance, with a validation F1-score of 94% and IoU of 90.78%. Besides, the morphometric analysis that shows strong alignment between manual annotations and automated segmentation with nerve length and tortuosity closely matching manual measurements. Furthermore, our analysis includes a comprehensive assessment of axon density and morphological features across different CAPRA-S prostate cancer risk categories revealing a significant decline in axon density correlating with higher CAPRA-S prostate cancer risk scores. Our paper suggests the potential utility of neuronal markers in the prognostic assessment of prostate cancer in aiding the pathologist's assessment of tumor sections and advancing our understanding of neurosignaling in the tumor microenvironment.

New Classification of Tumor Microvessels and the Risk of Regional Metastasis in Squamous Cell and Glandular Cancers

Previously, we proposed a new classification of tumor microvessels (MVs) on the basis of their morphological features and clinical significance. The aim of this study was to summarize the obtained results and establish the predictive value of different types of tumor MVs for assessing the risk of metastasis to regional lymph nodes (RLNs) in glandular and squamous cell carcinomas.<i> Materials and methods.</i> A total of 385 archival samples of gastric cancer stages I--III, breast cancer stages I--IIIA, cervical squamous cell carcinoma stages I--IIA, and lung squamous cell carcinoma stages I--IIIA were studied. The tumor sections were processed routinely and subjected to immunohistochemistry with antibodies against cluster of differentiation 34 (CD34) and podoplanin. To assess independent predictors of the risk of metastasis to RLNs, correlation analysis and univariate and multivariate logistic regression analyses were performed. Statistical analysis was performed via Statistica 10.0 software. <i>Results.</i> For patients with gastric cancer and breast cancer, the independent predictors of a high risk of metastasis to RLNs are T2 (p=0.007) and T3 (p<0.00001) stages, tumor grade 3 (p=0.0002), the presence of lymphovascular invasion (LVI) (p=0.044) and peritumoral retraction clefts (p=0.008). For patients with squamous cell carcinoma of the cervix and lung, independent predictors of a high risk of metastasis to RLNs are the T2 (p=0.01) and T3 (p=0.007) stages, the presence of LVI (p=0.0014), dilated capillaries (DCs) of the "contact type" (p=0.0007), capillaries in the tumor solid component (p=0.046) and peritumoral retraction cleftings (p=0.0006). <i>Conclusion</i>. The results of the present study indicate that when assessing the risk of metastasis to RLNs, it is advisable to consider the presence of peritumoral retraction clefting and LVI in both glandular and squamous cell carcinomas. In addition, in squamous cell carcinomas, the accuracy of assessing the risk of metastasis to RLNs can be increased by taking into account "contact-type" DCs and capillaries in the solid component of the tumor.

Nuclear pleomorphism in canine cutaneous mast cell tumors: Comparison of reproducibility and prognostic relevance between estimates, manual morphometry, and algorithmic morphometry.

Variation in nuclear size and shape is an important criterion of malignancy for many tumor types; however, categorical estimates by pathologists have poor reproducibility. Measurements of nuclear characteristics can improve reproducibility, but current manual methods are time-consuming. The aim of this study was to explore the limitations of estimates and develop alternative morphometric solutions for canine cutaneous mast cell tumors (ccMCTs). We assessed the following nuclear evaluation methods for accuracy, reproducibility, and prognostic utility: (1) anisokaryosis estimates by 11 pathologists; (2) gold standard manual morphometry of at least 100 nuclei; (3) practicable manual morphometry with stratified sampling of 12 nuclei by 9 pathologists; and (4) automated morphometry using deep learning-based segmentation. The study included 96 ccMCTs with available outcome information. Inter-rater reproducibility of anisokaryosis estimates was low (k = 0.226), whereas it was good (intraclass correlation = 0.654) for practicable morphometry of the standard deviation (SD) of nuclear size. As compared with gold standard manual morphometry (area under the ROC curve [AUC] = 0.839, 95% confidence interval [CI] = 0.701-0.977), the prognostic value (tumor-specific survival) of SDs of nuclear area for practicable manual morphometry and automated morphometry were high with an AUC of 0.868 (95% CI = 0.737-0.991) and 0.943 (95% CI = 0.889-0.996), respectively. This study supports the use of manual morphometry with stratified sampling of 12 nuclei and algorithmic morphometry to overcome the poor reproducibility of estimates. Further studies are needed to validate our findings, determine inter-algorithmic reproducibility and algorithmic robustness, and explore tumor heterogeneity of nuclear features in entire tumor sections.

Abstract A033: Integrating spatial transcriptomics and second harmonic generation imaging to identify targets of supermere-associated Discoidin Domain Receptor 1 involved in collagen alignment

Abstract Immune checkpoint blockade has been shown to be an effective treatment for colorectal cancers (CRCs) highly infiltrated by CD8+ T cells. However, there is a paucity of CD8+ T-cell infiltration in the majority of CRCs. We previously identified the collagen-activated receptor Discoidin Domain Receptor 1 (DDR1) as a member of a four-gene signature associated with T-cell exclusion in CRC. The cleaved ectodomain (cECD) of DDR1 induces collagen alignment, leading to T-cell exclusion in a breast cancer model. We found that almost all DDR1 cECD is found in supermeres, a novel secreted nanoparticle identified by our lab, and that DDR1 cECD is required for supermere signaling in recipient cells. We hypothesized that supermere-associated DDR1 cECD signals to CRC stromal cells, leading to collagen alignment. To test this hypothesis, we developed a method to determine which genes are associated with collagen alignment and DDR1 expression. To do this, 30 μm thick CRC sections are imaged using a Nikon AX multiphoton microscope by second harmonic generation imaging, which permits label-free imaging of collagen fibers. Spatial heatmaps of the collagen alignment are generated from the resulting images. Spatial transcriptomics is performed on a serial tumor section using the Visium v2 platform and the gene expression matrix is integrated with the collagen alignment heatmap data. As proof-of-principle, two CRC tumors have been analyzed using this method. Highly aligned regions were found to be associated with expression of genes involved in extracellular matrix organization, keloidal collagen and specific fibroblast subtypes. Genes which may be regulated by DDR1 cECD and be involved in collagen alignment will be confirmed at the protein level by multiplexed immunofluorescence on serial sections and in vitro experiments. This method will provide a rich dataset to elucidate the roles of supermeres and DDR1 cECD in T-cell exclusion and collagen fiber alignment and may support the discovery of new strategies to overcome immune exclusion in CRC. Citation Format: Maxwell S. Hamilton, Oleg Tutanov, Harsimran Kaur, Alan J. Simmons, Clark Massick, Kasey Vickers, Ken S. Lau, Ambra Pozzi, Robert J. Coffey. Integrating spatial transcriptomics and second harmonic generation imaging to identify targets of supermere-associated Discoidin Domain Receptor 1 involved in collagen alignment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A033.

Pharmacokinetic Positron Emission Tomography Imaging of an Optimized CD38-Targeted 68Ga-Labeled Peptide in Multiple Myeloma: A Pilot Study.

Multiple myeloma (MM) is an incurable disease characterized by its clinical and prognostic heterogeneity. Despite conventional chemotherapy and autologous hematopoietic stem cell transplantation, the management of relapsed and refractory MM disease poses significant challenges, both medically and socioeconomically. CD38, highly expressed on the surface of MM cells, serves as a distinct tumor biological target in MM. Peptides offer advantages over antibodies, enabling precise tumor imaging and facilitating early tumor diagnosis and dynamic immunotherapy monitoring. In this study, we developed PF381, a CD38-targeted peptide, and investigated its role in diagnosis, biodistribution, and dosimetry through 68Ga-labeling for preclinical evaluation in tumor-bearing models. We screened a microchip-based combinatorial chemistry peptide library to obtain the amino acid sequence of PF381. Affinity for human CD38 was evaluated by SPRi. PF381 was conjugated with DOTA for radiolabeling with 68Ga, and the complex was characterized by HPLC. PET imaging was performed in murine tumor models after the administration of [68Ga]Ga-DOTA-PF381. Biodistribution analysis compared CD38-positive H929 and CD38-negative U266 tumors, and human radiation dosimetry was estimated. Tumor sections were stained for CD38 expression. SPRi showed that PF381 had a high affinity for CD38 with a KD of 2.49 × 10-8 M. HPLC measured a radiolabeling efficiency of 78.45 ± 7.91% for [68Ga]Ga-DOTA-PF381, with >98% radiochemical purity. PET imaging revealed rapid and persistent accumulation of radioactivity in CD38-positive H929 tumors, contrasting with negligible uptake in CD38-negative U266 tumors. Biodistribution confirmed higher uptake in H929 tumors (0.75 ± 0.03%ID/g) vs U266 (0.26 ± 0.08%ID/g, P < 0.001). The kidney received the highest radiation dose (3.57 × 10-02 mSv/MBq), with an effective dose of 1.41 × 10-02 mSv/MBq. Immunofluorescence imaging supported PET and biodistribution findings. We developed a novel peptide targeting CD38 and proved that 68Ga-labeled PF381 had rapid targeting and good tumor penetration capabilities. Therefore, 68Ga-labeled PF381 could achieve high sensitivity in vivo imaging for CD38-positive hematological malignancies.

Abstract B048: Microfluidic isolation of immune cell-derived extracellular vesicles in head and neck cancer patients on immune-checkpoint blockade

Abstract Background: Immune-checkpoint blockade (ICB), with or without chemotherapy, is the standard of care for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients get clinical benefit from it. The combined expression of PD-L1 in a tumor section is currently the only biomarker approved for response prediction. Yet, its invasive nature and suboptimal predictive performance underscores the need for new, non- invasive surrogates of immune activity in patients undergoing ICB. Extracellular vesicles (EVs) carry key molecules critical for cell-cell communication and have the potential to be used as a non-invasive, real-time biomarker of systemic immune activity, essential for success in ICB- based therapy. Methods: We used a multichannel, microfluidic device that employs an immune- affinity approach, ‘herringbone chip’ (EVHB-Chip), to retrospectively isolate EVs from twelve HNSCC patients before and after the first cycle of ICB. One mL of plasma was used per patient per time point. For each patient, two devices were serially used, with each sample continuously following through each chip in a non-destructive manner—the first device aimed to isolate B cell EVs (antibodies against CD19 and CD20). The second device was optimized to capture T cell EVs (against CD274/PD-L1). Digital-droplet PCR was performed after EVs were isolated to detect the RNA transcripts in the EV cargo. Serial sampling was performed for six out of the twelve patients. Response assessment was conducted via RECIST1.1 criteria or clinical evaluation. Results: Most of our cohort was male 7/12 (57%) and had oral cavity 4/12 (33%) or oropharynx 4/12 (33%) tumors. Additionally, 11/12 (91%) patients received ICB monotherapy. Most of the cohort had a PD-L1 ≤ 1 (7/12; 58%), 7/12 patients experienced disease progression (PD), 4/12 experienced stable disease (SD), and one patient had a partial response (PR). Most of the RNA signal in pre-ICB samples was obtained from CD20 (12/12; 100%), followed by CD45 (10/12; 83%), CD19 (9/12; 75%), and CD274/PD-L1 (5/12; 41%). No difference was found in the RNA signal pre-ICB between responders and non-responders (p=0.1). For the patients with serial sampling available, the signal from CD274/PD-L1 increased after one infusion of ICB in 4/6 patients and remained negative in one. A trend towards a high amount of T cell-derived EVs was observed for the entire cohort (p=0.07; p=0.08) when comparing the amount of RNA via our EV marker (GAPDH, ACTB) between T and B cell devices. Patients with higher than 11.4/mL copies of CD19 (median value for the cohort) in the pre-ICB plasma sample tended to have a higher overall survival (HR 0.26, 95% CI: 0.06-1.13; p-value: 0.05) and progression-free survival (HR 0.28, 95% CI: 0.07-1.16; p-value: 0.06). Conclusion: The EVHB-Chip allowed for the detection and profiling of T and B cell-derived EVs in pre-ICB plasma samples of HNSCC patients undergoing ICB in this pilot cohort. Higher CD19 signal pre-ICB could potentially impact response and survival outcomes in ICB-based therapies. Citation Format: Andrew R. Levy, Daniel A Ruiz Torres, Uma Paithankar, Raheel Ahmad, Daniel C Rabe, Daniel L Faden, Shannon L Stott. Microfluidic isolation of immune cell-derived extracellular vesicles in head and neck cancer patients on immune-checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B048.

Automated Quantification of Tertiary Lymphoid Structures in Human Tumor Samples Using Immunofluorescence and AI-Powered Analysis Pipeline.

The tumor microenvironment (TME) is a complex entity comprising not only tumor cells but also immune, stromal, and endothelial cells. Preclinical and clinical studies indicate that the density, localization, function, and organization of immune infiltration can influence survival probability and treatment response in many cancers. Among these cell organizations, the clustering of T and B cells into tertiary lymphoid structures (TLS) has been associated with favorable clinical outcomes. In this protocol, we propose a protocol for 7-plex immunofluorescence staining for identifying six cell types enriched in TLS in formalin-fixed paraffin-embedded (FFPE) human tumor sections. Additionally, we provide a detailed methodology for quantifying these cell subtypes using the Halo-AI analysis software. This approach will enable a more precise and detailed characterization of TLS in the TME, opening new avenues for understanding their role in anticancer treatment response.

Neuroendocrine differentiation and serotonin expression in oesophageal adenocarcinomas after neoadjuvant therapy: correlation with clinicopathological features and outcome.

Oesophageal adenocarcinoma (EAC) is a glandular or mucinous epithelial malignancy that can show immunohistochemical evidence of neuroendocrine differentiation (NED) and express the hormone serotonin. The objective of this study was to correlate the presence of NED and serotonin with clinicopathological characteristics and patient outcome after neoadjuvant chemoradiation. A retrospective cohort of patients treated between 2002 and 2021 was established and included 218 oesophagectomy specimens with residual tumour. Representative full-face sections of tumour were stained for synaptophysin, chromogranin-A and serotonin by immunohistochemistry, and staining results were correlated with disease-free survival (DFS) and overall survival (OS). In total, 129 (59%) tumours showed evidence of NED, defined as immunohistochemical expression of synaptophysin or chromogranin-A, while 40 (18%) showed evidence of NED and expressed serotonin. Patients with neuroendocrine-positive tumours had significantly shorter median OS compared to those with neuroendocrine-negative tumours (22.5 versus 48.8 months, P = 0.006), but similar median DFS (13.3 versus 17.8 months, P = 0.34). Using Cox regression, the association between NED and OS was significant in univariate [hazard ratio (HR) = 1.68, 95% confidence interval (CI) = 1.16-2.45] and multivariate (HR = 1.65, 95% CI = 1.08-2.52) analysis. Patients with serotonin-expressing tumours had similar median OS (21.7 versus 25.9 months, P = 0.24) and DFS (7.3 versus 15.6 months, P = 0.12) compared to those with NED but lacking serotonin. Using Cox regression, serotonin expression was associated with reduced OS in univariate (HR = 1.62, 95% CI = 1.06-2.47) but not multivariate (HR = 1.03, 95% CI = 0.64-1.65) analysis. Our findings support NED as independent predictor of OS in EAC after neoadjuvant chemoradiation. While a subset of tumours with NED expressed serotonin, this did not provide additional prognostic information.

Assessment of Tumor Budding in Colorectal Carcinoma by Cytokeratin Staining

Background: Tumor budding has been receiving increasing recognition in many studies, as an important independent prognostic factor in colorectal carcinoma (CRC). In histopathological reporting of CRC, it is defined as the presence of single tumor cell or small clusters of up to 4 cells in the advancing edge of the tumor stroma.Objectives: We aim to determine the frequency of tumor budding by cytokeratin staining, in relation to the depth of invasion, lymph node involvement, perineural invasion and tumor grade in colorectal carcinoma.MaterialS and Methods: This descriptive cross sectional study was carried out from November 2022 till April 2023. During these six months, 62 hemi-colectomy specimens of colorectal carcinoma, received in histopathology lab were included in this study. The samples belonged to the patients who were biopsy proven cases of colorectal carcinoma and those who were not given any pre-operative therapy. The age range was from 20 to 80 years including both genders. After processing the tumor sections were examined under microscope, on both conventional stains and the Pan-Cytokeratin (AE1/AE3) stain. The results were interpreted by consultant histopathologists for tumor budding score. Tumor buds less than 10 per high power field was considered as low score. 10 and more buds per high power field were considered as high score.Results: When tumor grade and depth of invasion was compared to tumor budding score, the comparison was not statistically significant with p value of 0.06 and 0.937 respectively, whereas its relationship with lymph node involvement and perineural invasion was found to be statistically highly significant with p value of &lt; 0.001.Conclusion: This study concluded that tumor budding has a statistically significant relationship with lymph node metastasis and perineural invasion, but not with tumor grade and depth of invasion (T stage).Keywords: Colorectal carcinoma, Drade, Lymph node, Pan-cytokeratin, Perineural invasion, Stage.

CSIG-04. ANTI-TUMOR RESPONSE OF ONCOMAGNETIC MONOTHERAPY IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most lethal primary malignant brain tumor with a median survival of 15–20 months. Concurrent temozolomide (TMZ) chemotherapy and radiation (XRT) remain the current standard of care (SOC) treatment for newly diagnosed GBM. The addition of tumor treating fields (TTFs) improves median survival for newly diagnosed GBM from 16 to 20.9 months. This modest improvement underscores the urgent need to identify a new treatment modality as a safe and effective therapy. PURPOSE The noninvasive Oncomagnetic device developed in our laboratory selectively kills glioblastoma (GBM) in vitro. We studied the cellular and molecular effects of Oncomagnetic monotherapy (OMT) on GBM cell lines and investigated immune response in a syngeneic mouse model. RESULTS OMT significantly reduces cell proliferation and cell survival in vitro. OMT induces persistent ROS primarily by increasing superoxide level in cancer cells and reduces mitochondrial-membrane potential in GBM cells. It also induces DNA damage and arrests cells in G1-phase of the cell cycle. Whole-body Oncomagnetic stimulation caused a substantial retardation of tumor growth and reduction of the contrast-enhanced tumor volume in 9.4T MRI scans. OMT showed significant increase in overall survival in treated mice. We also observed upregulated immune response indicated by RNA sequencing data obtained from OMT-treated GBM cells as well as in treated GBM mouse tumor sections by imaging mass cytometry analysis. The tSNE analysis of various immune clusters suggests significant increase in immune response by activating CD4+, CD8+, murine-macrophages and M1-macrophages located at tumor site in the treated group. CONCLUSION The obtained data indicate significant anti-tumor response by inhibiting cancer signaling pathways and an increased immune response. Our findings suggest unique mechanism of action for OMT stimulation and provide a strong rationale for standalone OMT for GBM.

Broussoflavonol F exhibited anti-proliferative and anti-angiogenesis effects in colon cancer via modulation of the HER2-RAS-MEK-ERK pathway

BackgroundA prenylated flavonoid, broussoflavonol F (BFF), was isolated from Macaranga genus with cytotoxicities against various cancer cells, though its underlying mechanisms have not been fully elucidated. HypothesisThis study aimed to investigate the anti-tumor and anti-angiogenesis activities of BFF and its underlying mechanisms in colon cancer. MethodIn the in vitro study, the cytotoxic effects of BFF in human colon cancer HCT-116 and LoVo cells were examined using MTT assay, BrdU assay and colony formation assay. The anti-proliferative effects of BFF in these cells were assessed via cell apoptosis and cell cycle analysis using flow cytometry. The anti-angiogenesis effects of BFF in human endothelial HEMC-1 cells were also detected using scratch wound healing assay and tube formation assay. While the in vivo effects of BFF in colon cancer were further examined in zebrafish embryos and HCT116 tumor-bearing mice. The underlying mechanisms of BFF were predicted using network pharmacology analysis, and Western blotting was performed to validate both in vitro and in vivo results. ResultsBFF exhibited cytotoxicities on 5 colon cancer cell lines, as well as anti-proliferative activities via inducing apoptosis and cell cycle arrest at the G0/G1 phase in HCT116 and LoVo cells. BFF at 1.25–5 µM also suppressed cell proliferation in these two colon cancer cell lines by downregulating HER2, RAS, p-BRAF, p-MEK and p-Erk protein expressions. In addition, BFF at 2.5–5 µM could significantly decrease the length of subintestinal vessels of zebrafish embryos through decreasing mRNA expressions of NRP1a, PDGFba, PDGFRb, KDR, FLT1 and VEGRaa. Besides, BFF exhibited anti-angiogenesis activity via inhibiting cell proliferation, motility and tube formation in HMEC-1 cells. Furthermore, intraperitoneal administration of BFF (10 mg/kg) suppressed tumor growth and decreased the expression of tumor proliferation marker Ki-67 and angiogenesis marker CD31 in the tumor tissues in HCT116 tumor-bearing mice. BFF treatment could also significantly decrease expressions of RAS, p-BRAF, p-MEK and p-Erk in the tumor section, which are consistent with the in vitro results. ConclusionsThis study revealed the anti-tumor and anti-angiogenesis effects of BFF in colon cancer. This is the first report of the in vitro and in vivo anti-proliferative activity of BFF in colon cancer through regulating the HER2-RAS-MEK-ERK pathway. These findings further support the research development of BFF as an anti-cancer agent in colon cancer.