Necrotic Tumor Cells Research Articles

Tumor growth and vascular redistribution contributes to the dosimetric preferential effect of microbeam radiotherapy: a Monte Carlo study

The radiobiological mechanisms behind the favorable response of tissues to microbeam radiation therapy (MRT) are not fully described yet. Among other factors, the differential action to tumor and normal tissue vasculature is considered to contribute to MRT efficacy. This computational study evaluates the relevance of tumor growth stage and associated vascular redistribution to this effect. A multiscale approach was employed with two simulation softwares: TOPAS and CompuCell3D. Segmentation images of the angioarchitecture of a non-bearing tumor mouse brain were used. The tumor vasculature at different tumor growth stages was obtained by simulating the tumor proliferation and spatial vascular redistribution. The radiation-induced damage to vascular cells and consequent change in oxygen perfusion were simulated for normal and tumor tissues. The multiscale model showed that oxygen perfusion to tissues and vessels decreased as a function of the tumor proliferation stage, and with the decrease in uniformity of the vasculature spatial distribution in the tumor tissue. This led to an increase in the fraction of hypoxic (up to 60%) and necrotic (10%) tumor cells at advanced tumor stages, whereas normal tissues remained normoxic. These results showed that tumor stage and spatial vascular distribution contribute to the preferential effect of MRT in tumors.

Evaluation of Vincamine Loaded with Silver Nanoparticles as a New Potential Therapeutic Agent Against Ehrlich's Solid Carcinoma in Mice.

Vincamine, a monoterpenoid indole alkaloid with vasodilatory properties, is extracted from the leaves of Vinca minor. The present study aimed to determine the potential anticancer effects of vincamine loaded in silver nanoparticles (VCN-AgNPs) in mice with Ehrlich solid carcinoma (ESC). After tumor transplantation, the mice were divided into five groups: ESC, ESC+Cisplatin (CPN; 5 mg/kg), ESC+VCN (40 mg/kg), ESC+AgNPs (6 mg/kg), and ESC+VCN-AgNPs (20 mg/kg). The administration of VCN-AgNPs to ESC-bearing mice improved their survival rate and reduced their body weight, tumor size, and tumor weight compared to the ESC group. Furthermore, VCN-AgNPs intensified oxidative stress in tumor tissues, as evidenced by elevated levels of lipid peroxidation (LPO) and nitric oxide (NO), along with a reduction in the levels of the antioxidants investigated (GSH, GPx, GR, SOD, CAT, and TAC). Furthermore, VCN-AgNPs increased the apoptotic proteins Bax and caspase-3, decreased the anti-apoptotic protein (Bcl-2), increased the inflammatory markers TNF-α and IL-1β, and inhibited angiogenesis by lowering VEGF levels in tumor tissues, all of which led to apoptosis. Furthermore, histopathological studies showed that VCN-AgNPs suppressed the progression of Ehrlich carcinoma and induced the formation of clusters of necrotic and fragmented tumor cells. VCN-AgNPs possess cytotoxic and genotoxic effects against ESC because of their pro-oxidant, pro-apoptotic, pro-inflammatory, and antiangiogenic effects. Additionally, the combination of VCN-AgNPs was more effective and safer than chemically synthesized AgNPs, as indicated by an increase in the lifespan of animals and the total tumor inhibition index.

An approach for improvement of the accuracy of cancer gene panel testing.

Tissue-based comprehensive genomic profiling (CGP) is increasingly being employed for genotype-directed therapies in patients with advanced cancer. However, tissue availability may limit their potential applications. In Japan, the cost of cancer gene panel tests is covered by public insurance for patients diagnosed with advanced solid tumors once in their lifetime. Therefore, it is essential to improve the success rate (reportability) and accuracy of CGP tests. The purpose of this study was to identify the factors associated with efficient and accurate CGP testing using relevant information obtained from real-world data. This study included 159 samples analyzed using tumor-only panel FoundationOne® CDx cancer genome profiling (F1CDx) and 85 samples analyzed using matched-pair panel OncoGuide™ NCC Oncopanel system (NCCOP) at St. Marianna University Hospital. Sample characteristics (fixation conditions, storage period, histology, tumor cell ratio, and genomic tumor cell content), CGP performance, and quality control status were evaluated across all 244 tested samples. In 237/244 samples (97.1%), CGP testing results were successfully obtained [F1CDx, 99.4% (158/159) and NCCOP, 92.9% (79/85)]. An increased number of fibroblasts, inflammatory cells, and necrotic tumor cells, long-term storage, and/or prolonged fixation of tissue sections were involved in the unreported results and/or qualified CGP results. In addition, a negative correlation between median insert size values and ΔΔCq was observed in the NCCOP system. We identified various factors associated with efficient and accurate CGP testing using relevant information obtained from real-world data, suggesting that thorough selection and preparation of tissue sections could optimize CGP and maximize useful information.

Validation of Circulating Tumor DNA As an Alternative to Tumor Tissue Biopsy in Genotyping across Multiple Lymphoma Subtypes

Introduction Genotyping plays an important role in the diagnosis, risk stratification and treatment response surveillance of lymphoma, a disease with considerable heterogeneity in clinical and pathological characteristics. To date, tumor tissue biopsy based genotyping is still considered as gold standard in lymphoma, but its application is hampered by the invasive nature and imperfect performance in capturing spatial and temporal tumor heterogeneity. Circulating tumor DNA (ctDNA), which is shed into peripheral blood by apoptotic or necrotic tumor cells, has shown the potential as a noninvasive alternative to tumor tissue DNA (tDNA) for molecular profiling. Many efforts have been made to investigate the feasibility of ctDNA based on next-generation sequencing (NGS) in lymphoma, but most of the studies were conducted in Caucasian population and limited to B-cell lymphoma patients. In the present study, we evaluated the performance of plasma-derived ctDNA against tDNA based genotyping across multiple lymphoma subtypes. Methods Between 4/2021 and 5/2023, a total of 110 patients were enrolled in this study. Matched tumor tissue biopsies and peripheral blood were collected at diagnosis. Both tDNA and ctDNA were extracted and subjected to a targeted capture NGS assay covering the entire coding region or recurrently mutated region of 196 genes. The mean coverage depth was 2431x and 2513x for tDNA and ctDNA, respectively. The limit of detection (LOD) was 1% and 0.5% for tDNA and ctDNA, respectively. Results The enrolled cohort comprised of 63 (57.3%) male and 47(42.7%) female patients, with a median age of 58 years old (range 1.25-89). Among the 110 patients, the most prevalent subtype is diffuse large B-cell lymphoma (DLBCL，n=45), following by angioimmunoblastic T-cell lymphoma (AITL, n=16), anaplastic large cell lymphoma (ALCL, n=13) , mantle cell lymphoma (MCL, n=10), follicular lymphoma (FL, n=9), primary mediastinal large B-cell lymphoma (PMBL, n=9), extranodal natural killer/T cell lymphoma (ENKTL, n=4), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS, n=4). For further analysis, DLBCL, MCL, FL and PMBL were categorized as B-cell lymphoma group (n=73), while AITL, ALCL, ENKTL and PTCL-NOS were categorized as T-cell lymphoma group (n=37). Mutations were detected in 109（99.10%）tDNA and 110（100%）ctDNA, respectively. A total of 1586 mutations involving 177 genes were detected in tDNA, and the median number of mutations per sample was 12 (range 0-62), with KMT2D (39.09%), KMT2C (30.90%), IGLL5 (30.00%), TET2 (25.45%) and TP53 (20.90%) being the top 5 mutated genes. For ctDNA, a total of 1750 mutations involving 178 genes were detected and the median number of mutations per sample was 14 (range 2-63), with KMT2D (37.27%), KMT2C (29.09%), IGLL5 (27.27%), TET2 (25.45%) and DNMT3A (27.27%) being the top 5 mutated genes. To determine the concordance of mutations detected in tDNA and ctDNA, sensitivity was calculated utilizing tDNA as gold standard. The overall sensitivity of ctDNA based genotyping was 85.39% (95%CI: 82.36%-88.41%), with B-cell lymphoma showing a slightly higher sensitivity than T-cell lymphoma (86.17%, 95%CI: 83.51%-88.83% vs. 83.84%, 95%CI: 76.30%-91.32%). In terms of subtypes, PTCL-NOS exhibited the highest sensitivity (89.58%, 95%CI: 56.43%-122.73%, while AITL exhibited the lowest sensitivity (78.53%, 95%CI: 64.09%-92.97%) (Figure 1A). In addition, the difference of mutational spectrum between B-cell lymphoma and T-cell lymphoma was precisely depicted by ctDNA based genotyping. We discovered mutations in TET2, RHOA, and PLCG1 were significantly present in T-cell lymphoma, while mutations in MYD88, CD79B, CARD11, CREBBP, MYC, PIM1, IGLL5, ATM, SOCS1 and HIST1HIE were significantly enriched in B-cell lymphoma (Figure 1B). Conclusion In the present study, recurrent mutations across a variety of lymphoma subtypes were identified by ctDNA based genotyping, which were in high concordance with tDNA based genotyping. These data demonstrated that ctDNA based genotyping is a reliable noninvasive alternative to tumor tissue biopsy for molecular profiling of lymphoma patients, especially for patients whose tissues are not accessible for genotyping.

Tumor cell–derived spermidine is an oncometabolite that suppresses TCR clustering for intratumoral CD8+ T cell activation

The activation and expansion of T cells that recognize cancer cells is an essential aspect to antitumor immunity. Tumors may escape destruction by the immune system through ectopic expression of inhibitory immune ligands typically exemplified by the PD-L1/PD-1 pathway. Here, we reveal another facet of tumor evasion from T cell surveillance. By secretome profiling of necrotic tumor cells, we identified an oncometabolite spermidine as a unique inhibitor of T cell receptor (TCR) signaling. Mechanistically, spermidine causes the downregulation of the plasma membrane cholesterol levels, resulting in the suppression of TCR clustering. Using syngeneic mouse models, we show that spermidine is abundantly detected in the tumor immune microenvironment (TIME) and that administration of the polyamine synthesis inhibitor effectively enhanced CD8+ T cell-dependent antitumor responses. Further, the combination of the polyamine synthesis inhibitor with anti-PD-1 immune checkpoint antibody resulted in a much stronger antitumor immune response. This study reveals an aspect of immunosuppressive TIME, wherein spermidine functions as a metabolic T cell checkpoint that may offer a unique approach for promoting tumor immunotherapy.

Combination of immune checkpoint blockade and targeted gene regulation of angiogenesis for facilitating antitumor immunotherapy.

The rapid development of tumor immunotherapy has improved the management of patients with cancer. However, several key problems of tumor immunotherapy, including the insufficient activation of effector T cells, poor tumor invasion, and poor immune killing ability, lead to a low response rate. In the present study, a synergistic strategy was developed by combining in situ tumor vaccines, gene-mediated downregulation of tumor angiogenesis, and anti-PD-L1 therapy. In situ tumor vaccines and antitumor angiogenesis were achieved by codelivering unmethylated cytosine-phosphate-guanine (CpG) and vascular endothelial growth factor (VEGF)-silencing gene (shVEGF) via a hyaluronic acid (HA)-modified HA/PEI/shVEGF/CpG system. Necrotic tumor cells and CpG adjuvants formed in situ tumor vaccines and activated the host immune response. Moreover, VEGF silencing reduced tumor angiogenesis and prompted the homogeneous distribution of tumor blood vessels to facilitate immune cell infiltration. Meanwhile, anti-angiogenesis also improved the immunosuppressive tumor microenvironment. To further improve the specific tumor-killing effect, an anti-PD-L1 antibody was introduced for immune checkpoint blockade, thereby boosting antitumor immune responses. The combination therapy strategy presented in the present study could act in the multiple stages of the tumor immunotherapy cycle, which is expected to offer a new avenue for clinical tumor immunotherapy.

Tumor‐targeted nano‐adjuvants to synergize photomediated immunotherapy enhanced antitumor immunity

AbstractPhotomediated immunotherapy explored to combine the anti‐cancer effect of phototherapy with the immune enhancement ability of immunotherapy, and shown great prospects for cancer treatment strategies. However, photomediated immunotherapy triggered antitumor immunity through the release of tumor antigens and damage‐associated molecular patterns from necrotic tumor cells was not enough mighty to improve the therapeutic benefits due to the immunosuppressive tumor microenvironments. Herein, a tumor‐targeted nano‐adjuvant Fucoidan@Al(OH)3‐Poly(I:C)/IR‐820 for tumor‐targeted therapy and metastasis inhibition was designed and prepared. The intrinsic immunomodulatory effects of tumor‐targeted nano‐adjuvant and their ability to simultaneously trigger tumor antigen release, thereby reversing immunosuppression and achieving potent antitumor immunity and augmented cancer therapy, were explored. The results proved that the multifunctional nano‐adjuvant combined with photothermal, photodynamic, and immunotherapy could effectively treat breast cancer and had metastasis inhibition effect by enhancing anti‐tumor immunity through immunomodulation, it should have great application potential in the treatment of breast cancer.

Preclinical PET Imaging of Tumor Cell Death following Therapy Using Gallium-68-Labeled C2Am

There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

Lactate oxidase/vSIRPα conjugates efficiently consume tumor-produced lactates and locally produce tumor-necrotic H2O2 to suppress tumor growth

Aggressive tumor formation often leads to excessive anaerobic glycolysis and massive production and accumulation of lactate in the tumor microenvironment (TME). To significantly curb lactate accumulation in TME, in this study, lactate oxidase (LOX) was used as a potential therapeutic enzyme and signal regulatory protein α variant (vSIRPα) as a tumor cell targeting ligand. SpyCatcher protein and SpyTag peptide were genetically fused to LOX and vSIRPα, respectively, to form SC-LOX and ST-vSIRPα and tumor-targeting LOX/vSIRPα conjugates were constructed via a SpyCatcher/SpyTag protein ligation system. LOX/vSIRPα conjugates selectively bound to the CD47-overexpressing mouse melanoma B16-F10 cells and effectively consumed lactate produced by the B16-F10 cells, generating adequate amounts of hydrogen peroxide (H2O2), which induces drastic necrotic tumor cell death. Local treatments of B16-F10 tumor-bearing mice with LOX/vSIRPα conjugates significantly suppressed B16-F10 tumor growth in vivo without any severe side effects. Tumor-targeting vSIRPα may allow longer retention of LOX in tumor sites, effectively consuming surrounding lactate in TME and locally generating adequate amounts of cytotoxic H2O2 to suppress tumor growth. The approach restraining the local lactate concentration and H2O2 in TME using LOX and vSIRPα could offer new opportunities for developing enzyme/targeting ligand conjugate-based therapeutic tools for tumor treatment.

A phase-field model for non-small cell lung cancer under the effects of immunotherapy.

Formulating mathematical models that estimate tumor growth under therapy is vital for improving patient-specific treatment plans. In this context, we present our recent work on simulating non-small-scale cell lung cancer (NSCLC) in a simple, deterministic setting for two different patients receiving an immunotherapeutic treatment. At its core, our model consists of a Cahn-Hilliard-based phase-field model describing the evolution of proliferative and necrotic tumor cells. These are coupled to a simplified nutrient model that drives the growth of the proliferative cells and their decay into necrotic cells. The applied immunotherapy decreases the proliferative cell concentration. Here, we model the immunotherapeutic agent concentration in the entire lung over time by an ordinary differential equation (ODE). Finally, reaction terms provide a coupling between all these equations. By assuming spherical, symmetric tumor growth and constant nutrient inflow, we simplify this full 3D cancer simulation model to a reduced 1D model. We can then resort to patient data gathered from computed tomography (CT) scans over several years to calibrate our model. Our model covers the case in which the immunotherapy is successful and limits the tumor size, as well as the case predicting a sudden relapse, leading to exponential tumor growth. Finally, we move from the reduced model back to the full 3D cancer simulation in the lung tissue. Thereby, we demonstrate the predictive benefits that a more detailed patient-specific simulation including spatial information as a possible generalization within our framework could yield in the future.

Calpains Released from Necrotic Tumor Cells Enhance Antigen Cross-Presentation to Activate CD8+ T Cells In Vitro

The initiation of CD8+ T cell responses against dead cell-associated Ags is tightly regulated, facilitating adaptive immunity against pathogens and tumors while preventing autoimmunity. It is now well established that dying cells actively regulate the generation of CD8+ T cell responses via the release or exposure of damage-associated molecular patterns. However, it is unclear whether nonproteasomal proteases (activated in stressed and dying cells) can influence the availability of Ags for cross-presentation. Using a mouse model of immunogenic necrosis, we investigated the role of tumor-derived proteases in the priming of CD8+ T cells. We demonstrate that proteases released from necrotic tumor cells can degrade whole-protein Ag, generating proteolytic intermediates that are efficiently cross-presented by dendritic cells and enhance CD8+ T cell cross-priming. We identify a dominant role for calpain proteases, which are activated during necrotic cell death induced by severe heat shock. Mechanistically, proteolytic intermediates generated by tumor-derived proteases associate with necrotic tumor cell debris, which acts as a vehicle for Ag transfer that facilitates highly efficient cross-presentation in dendritic cells. Our results suggest that proteolytic systems activated in Ag donor cells during cell death may influence the availability of antigenic substrates for cross-presentation, thereby regulating the antigenicity of cell death.

Effect of temperature and duration of pasteurization exposure on tumor viability in osteosarcoma

This study aims to examine the effect of temperature and duration of pasteurization exposure on osteosarcoma tumor tissue degeneration, necrosis, and tumor cell viability. Using osteosarcoma tumor tissue, researchers evaluated degeneration, necrosis, and tumor cell viability following exposure to pasteurization. Hematoxylin-eosin (HE) staining was utilized to assess degeneration and necrosis, whereas CKAP4 immunohistochemical (IHC) staining was utilized to assess cell viability. The Kruskal-Wallis test showed a significant increase in degenerated tumor cells (p = 0.004) with the highest number found in the 60°C temperature group with a 40-minute exposure time, and a significant increase in necrotic tumor cells (p = 0.000) with the highest number found in the 70°C temperature group with a 40-minute exposure time. The 70°C for 40 minutes group had the fewest viable tumor cells (p = 0.000). This study found that the dose of pasteurization exposure had an effect on osteosarcoma cell degeneration, necrosis, and viability. With increasing temperature and duration of pasteurization, tumor cells exhibited increased degeneration and necrosis, as well as decreased viability, with the optimal exposure occurring at 70°C for 40 minutes, which could kill all tumor cells.

The Delivery of Extracellular "Danger" Signals to Cytosolic Sensors in Phagocytes.

Phagocytes, such as macrophages and dendritic cells, possess the ability to ingest large quantities of exogenous material into membrane-bound endocytic organelles such as macropinosomes and phagosomes. Typically, the ingested material, which consists of diverse macromolecules such as proteins and nucleic acids, is delivered to lysosomes where it is digested into smaller molecules like amino acids and nucleosides. These smaller molecules can then be exported out of the lysosomes by transmembrane transporters for incorporation into the cell’s metabolic pathways or for export from the cell. There are, however, exceptional instances when undigested macromolecules escape degradation and are instead delivered across the membrane of endocytic organelles into the cytosol of the phagocyte. For example, double stranded DNA, a damage associated molecular pattern shed by necrotic tumor cells, is endocytosed by phagocytes in the tumor microenvironment and delivered to the cytosol for detection by the cytosolic “danger” sensor cGAS. Other macromolecular “danger” signals including lipopolysaccharide, intact proteins, and peptidoglycans can also be actively transferred from within endocytic organelles to the cytosol. Despite the obvious biological importance of these processes, we know relatively little of how macromolecular “danger” signals are transferred across endocytic organelle membranes for detection by cytosolic sensors. Here we review the emerging evidence for the active cytosolic transfer of diverse macromolecular “danger” signals across endocytic organelle membranes. We will highlight developing trends and discuss the potential molecular mechanisms driving this emerging phenomenon.

In Vivo Evaluation of Gallium-68-Labeled IRDye800CW as a Necrosis Avid Contrast Agent in Solid Tumors.

Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [68Ga]Ga-DOTA-PEG4-IRDye800CW ([68Ga]Ga-1) with a radiochemical purity of >96% (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [68Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [68Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [68Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.

Tumoral Morphologic Features From Cervical Biopsies That Are Predictive of a Negligible Risk for Nodal Metastasis and Tumor Recurrence in Usual-type Cervical Adenocarcinomas: A Multi-institutional Study.

The metastatic or recurrent potential of localized human papillomavirus-associated endocervical adenocarcinoma (HPVA EAC) is difficult to predict, especially based upon biopsy alone. Recent analyses of small cohorts indicate that high tumor nuclear grade (TNG) and the presence of necrotic tumor debris (NTD) from HPVA EACs in cervical biopsy specimens are highly predictive of nodal metastasis (NM). In the present study, we aimed to investigate how reliably tumoral morphologic features from cervical biopsy specimens predict NM or tumor recurrence (TR) and patient outcomes in a large cohort of endocervical adenocarcinoma patients. A cohort comprised of 397 patients with HPVA EAC treated at 18 institutions was identified, and cervical biopsies were paired with their associated complete tumor resections for a total of 794 specimens. A variety of tumoral histologic features were examined for each paired specimen, including TNG (assessed on a 3-tiered scale of increasing abnormalities-TNG1, TNG2, TNG3) and NTD (defined by the presence of necrotic and apoptotic tumor cells within tumor glandular lumens admixed with granular and eosinophilic amorphous material and inflammatory cells), which were correlated with outcomes. The distribution of TNG in biopsies was as follows: 86 (21.7%) TNG1, 223 (56.2%) TNG2, and 88 (22.2%) TNG3. NTD was identified in 176 (44%) of the biopsy specimens. The sensitivity, specificity, positive predictive value, and negative predictive value of a TNG1 assignment in the biopsy being predictive of the same assignment in the full resection were 0.82 (95% confidence interval [CI]: 0.7-0.9), 0.895 (0.86-0.93), 0.593 (0.48-0.696), and 0.96 (0.94-0.98), respectively. Respective values for an NTD-negative status were 0.89 (95% CI: 0.83-0.92), 0.715 (0.64-0.77), 0.72 (0.65-0.77), and 0.89 (0.83-0.93), respectively. Compared with the other cases in each category, both TNG1 and an NTD-negative status were each significantly associated with lower rates of NM (odds ratio for TNG1=0.245, 95% CI: 0.070-0.857, P=0.0277; for NTD=0.199, 95% CI: 0.094-0.421, P<0.0001) and TR (odds ratio for TNG1=0.225, 95% CI: 0.051-0.987, P=0.0479; for NTD=0.367, 95% CI: 0.171-0.786, P=0.0099) independent of depth of stromal invasion, lymphovascular invasion, tumor size, FIGO stage, and Silva pattern. Overall, 73/379 (19%) cases were both TNG1 and NTD-negative on the biopsy, and none of these 73 cases showed NM (0%), but a single case (1.4%) showed TR. In contrast, among the 324 biopsies with TNG2/3 and/or presence of NTD, 62 (19.1%) had NM, and 41 (12.9%) had TR. In summary, 2 variables in combination (ie, TNG1 and NTD-negative) identified a subset of HPVA EAC patients-∼19%-with a 0% frequency of nodal metastases and only 1.4% frequency of recurrence. Biopsies highly but imperfectly predicted these features. Nonetheless, these findings may potentially be of clinical utility in the risk stratification of patients with HPVA EACs. This may allow some patients with a minimal risk of nodal metastases and TR to be identified at the biopsy phase, thereby facilitating more personalized, possibly less aggressive treatment.

Development and validation of a weakly supervised deep learning framework to predict the status of molecular pathways and key mutations in colorectal cancer from routine histology images: a retrospective study

SummaryBackgroundDetermining the status of molecular pathways and key mutations in colorectal cancer is crucial for optimal therapeutic decision making. We therefore aimed to develop a novel deep learning pipeline to predict the status of key molecular pathways and mutations from whole-slide images of haematoxylin and eosin-stained colorectal cancer slides as an alternative to current tests.MethodsIn this retrospective study, we used 502 diagnostic slides of primary colorectal tumours from 499 patients in The Cancer Genome Atlas colon and rectal cancer (TCGA-CRC-DX) cohort and developed a weakly supervised deep learning framework involving three separate convolutional neural network models. Whole-slide images were divided into equally sized tiles and model 1 (ResNet18) extracted tumour tiles from non-tumour tiles. These tumour tiles were inputted into model 2 (adapted ResNet34), trained by iterative draw and rank sampling to calculate a prediction score for each tile that represented the likelihood of a tile belonging to the molecular labels of high mutation density (vs low mutation density), microsatellite instability (vs microsatellite stability), chromosomal instability (vs genomic stability), CpG island methylator phenotype (CIMP)-high (vs CIMP-low), BRAFmut (vs BRAFWT), TP53mut (vs TP53WT), and KRASWT (vs KRASmut). These scores were used to identify the top-ranked titles from each slide, and model 3 (HoVer-Net) segmented and classified the different types of cell nuclei in these tiles. We calculated the area under the convex hull of the receiver operating characteristic curve (AUROC) as a model performance measure and compared our results with those of previously published methods.FindingsOur iterative draw and rank sampling method yielded mean AUROCs for the prediction of hypermutation (0·81 [SD 0·03] vs 0·71), microsatellite instability (0·86 [0·04] vs 0·74), chromosomal instability (0·83 [0·02] vs 0·73), BRAFmut (0·79 [0·01] vs 0·66), and TP53mut (0·73 [0·02] vs 0·64) in the TCGA-CRC-DX cohort that were higher than those from previously published methods, and an AUROC for KRASmut that was similar to previously reported methods (0·60 [SD 0·04] vs 0·60). Mean AUROC for predicting CIMP-high status was 0·79 (SD 0·05). We found high proportions of tumour-infiltrating lymphocytes and necrotic tumour cells to be associated with microsatellite instability, and high proportions of tumour-infiltrating lymphocytes and a low proportion of necrotic tumour cells to be associated with hypermutation.InterpretationAfter large-scale validation, our proposed algorithm for predicting clinically important mutations and molecular pathways, such as microsatellite instability, in colorectal cancer could be used to stratify patients for targeted therapies with potentially lower costs and quicker turnaround times than sequencing-based or immunohistochemistry-based approaches.FundingThe UK Medical Research Council.

Damage-associated molecular patterns and Toll-like receptors in the tumor immune microenvironment.

As clinically demonstrated by the success of immunotherapies to improve survival outcomes, tumors are known to gain a survival advantage by circumventing immune surveillance. A defining feature of this is the creation and maintenance of a tumor immune microenvironment (TIME) that directly and indirectly alters the host's immunologic signaling pathways through a variety of mechanisms. Tumor-intrinsic mechanisms that instruct the formation and maintenance of the TIME have been an area of intensive study, such as the identification and characterization of soluble factors actively and passively released by tumor cells that modulate immune cell function. In particular, damage-associated molecular pattern (DAMP) molecules typically released by necrotic tumor cells are recognized by innate immune receptors such as Toll-like receptors (TLRs) and stimulate immune cells within TIME. Given their broad and potent effects on the immune system, a better understanding for how DAMP and TLR interactions sculpt the TIME to favor tumor growth would identify new strategies and approaches for cancer immunotherapy.

Bone marrow trephine immunohistochemistry is useful in characterizing malignancy-associated myelonecrosis: A retrospective observational study.

We aim to describe the utility of immunohistochemistry (IHC) in characterizing malignancy-associated myelonecrosis (MN) on bone marrow trephine biopsies (BMBx) as a part of initial workup. Patten and intensity of antigenic immunoexpression in necrotic tumor cells on BMBx were evaluated in a series of cases using standardized avidin-biotin-complex immunoperoxidase technique after heat-induced epitope retrieval and compared the same with viable tumor cells wherever available. Fifteen out of 2494 (0.6%) cases (median age: 28years; range: 4 to 66years) had evidence of MN (extensive in eight, moderate in five, and focal in two) secondary to hematological (N=9) and solid (N=6) malignancies. Five (33.3%) had pancytopenia, and eight (53.3%) had difficult and/or hemodiluted aspirate. Antigenic expression for CD10, CD79a, CD3, CD7, and CD20 was retained by necrotic leukemic blasts or lymphoma cells; CD34, TdT, and PAX5 showed heterogeneous expression; and a weak Golgi zone (dot like) CD30 positivity was noted in Reed-Sternberg (RS) or RS-like giant cells. Necrotic epithelial metastases retained pancytokeratin in all and showed variable positivity for prostate-specific antigen, carcinoembryonic antigen, CK20, ER, PR, and GATA3. Necrotic neuroblastomas (N=2) retained positivity for synaptophysin and chromogranin, whereas retained nuclear positivity for NKX2.2 in necrotic Ewing family of tumor (N=1) aided in early diagnosis. Myelonecrosis may retain tumor antigenicity, and immunohistochemistry using selected panel of antibodies should be tried in such challenging cases for an early presumptive diagnosis and further decision making.

Abstract 1584: Efficacy and pharmacodynamic effect of anti-CD73/PD-L1 monoclonal antibodies in combination with chemotherapy: Observations from mouse tumor models

Abstract Intratumoral adenosine is a key immunosuppressive factor linked to poor prognosis and reduced efficacy of T cell checkpoint inhibitors. CD73 is an ectoenzyme and key node in the catabolic pathway responsible for sequential hydrolysis of extracellular ATP to adenosine. ATP is released from necrotic and damaged tumor cells; a phenomenon enhanced as consequence of cytotoxic chemotherapy or radiotherapy. A CD73 inhibiting human monoclonal IgG1-TM antibody, Oleclumab, is currently in phase 2 clinical development for treatment of patients with various solid tumors. The combination of CD73 inhibition with chemotherapy and T cell checkpoint inhibition was tested using two murine cancer models, CT26 (colorectal) or MCA205 (fibrosarcoma) implanted subcutaneously in BALB/c mice or C57BL/6 mice, respectively. Tumor bearing mice were treated with combinations of oxaliplatin and 5-fluorouracil and murine surrogate monoclonal antibodies for Oleclumab and Durvalumab (anti-PD-L1). CT26 implanted mice were also treated with the murine surrogate antibodies in the presence and absence of Docetaxel. In an attempt to define contribution of components, comparator groups received monotherapies and other iterations of the combination. Treatment with anti-CD73 and anti-PD-L1 antibodies, concomitantly with chemotherapy, resulted in improved tumor growth inhibition, plus increased proportions of complete tumor regression (P&amp;lt;0.05). FACS analysis of CT26 tumors highlighted elevated frequencies of intratumoral IFN-gamma secreting T and NK cells in the chemotherapy plus double IO combination group (P&amp;lt;0.05). Consistent with this, selective depletion of CD8 T cells significantly diminished tumor control and long term survival (P&amp;lt;0.05). RNAseq and Mass spectrometry based imaging techniques were utilized as tools to explore biomarker changes and mechanism. Citation Format: Jim Eyles, Amanda Watkins, Kristina Ilieva, Stef Mullins, Jude Anderton, Elena Galvani, Fabien Garcon, Kelli Ryan, Brajesh P. Kaistha, Andreas Dannhorn, Stephanie Ling, Tim Slidel, Gozde Kar, Alwin Schuller, Zachary A. Cooper, Kris Sachsenmeier, Nadia Luheshi, Rakesh Kumar, Robert W. Wilkinson. Efficacy and pharmacodynamic effect of anti-CD73/PD-L1 monoclonal antibodies in combination with chemotherapy: Observations from mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1584.

Abstract 1636: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering “don't eat me” and “don't find me” signals. Recent literature demonstrates that C-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) and macrophages (MPs) is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from the human tumor microenvironment, in particular, tumor-associated MPs. We evaluated whether CLEC-1 could also be a receptor for DAMPs and influences phagocytosis. We found that CLEC-1 fusion protein binds specifically to secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray), or culture stress conditions suggesting that ligands of CLEC-1 are generated upon stress and programmed cell death. Importantly, further to promising results in KO CLEC-1 mice, we observed in vivo that CLEC-1 deficient mice, in contrast to wild-type littermates, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy. Importantly, disruption of CLEC-1 signaling by Fc-CLEC-1 fusion protein also promotes tumor eradication. We then generated and identified different anti-human CLEC-1 antagonist monoclonal antibodies (mAbs) with the capacity to block CLEC-1/CLEC-1L interaction. We developed innovative antagonist CLEC-1 mAbs which, in contrast to non-antagonist CLEC-1 control mAb, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs or MPs. Indeed, TGFβ-polarized DCs phagocytose more efficiently a NSCLC cell line (A549) as well as Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) when CLEC-1 is antagonized by Abs. Similarly, macrophages significantly more efficiently engulfed human tumors in the presence of CLEC-1 antagonist Abs, in particular when tumor cells were opsonized such as Rituximab-opsonized Raji cells, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) or Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+). Importantly, we observed both in vitro and in vivo that DCs from Clec1a deficient mice cross-present more efficiently dead cell-associated antigens to CD8+ T cells (OT-1). We generated hCLEC-1 knock-in mice and in vivo preclinical evaluation of CLEC-1 blocking mAbs is ongoing. Altogether, these data illustrate that CLEC-1 broadly notably inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors, and hampers DC antigen cross-presentation. Citation Format: Vanessa Gauttier, Sabrina Pengam, Marion Drouin, Javier Saenz, Bérangère Evrard, Kevin Biteau, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Emmanuelle Wilhelm, Elise Chiffoleau, Nicolas Poirier. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1636.