Monitoring Tumor Response Research Articles

NALCN Promoter Methylation as a Biomarker for Metastatic Risk in a Cohort of Non-Small Cell Lung Cancer Patients

Liquid biopsy enables real-time monitoring of tumor development and response to therapy through the analysis of CTCs and ctDNA. NALCN is a sodium leak channel that is frequently involved in tumor evolution and immunity and acts as a tumor suppressor. Deletion of NALCN has been shown to increase cancer metastasis and the number of CTCs in peripheral blood. In this study, we investigated for the first time NALCN promoter methylation in (a) Aza-treated cell lines (A549, TE671, BT20, and MDA-MB-468), (b) paired NSCLC tissues (n = 22), and (c) plasma cell-free DNA (ctDNA) from patients with NSCLC (early stage n = 39, metastatic n = 39) and DNA from 10 healthy donors (HD) using a newly developed highly specific and sensitive real-time MSP method. Treatment with 5′-aza-dC induced the expression of NALCN only in the A549 cell line, suggesting that DNA methylation regulates its expression in certain cancers. The mRNA expression levels of NALCN were quantified in non-small cell lung cancer (NSCLC) and adjacent non-cancerous tissues, and it was found to be underexpressed in 54.5% of tumor tissues, with significantly higher expression in recurrence-free patients (p = 0.009) than in patients who relapsed. The NALCN methylation level was not statisticallysignificantlycorrelated with the corresponding expression (p = 0.439), while Kaplan–Meier analysis showed an association between NALCN promoter hypermethylation and worse disease-free intervals (DFIs) (p = 0.017). Evaluation of NALCN methylation in ctDNA revealed that it was detected in 5.1% of early and 10.2% of advanced cases. Our results strongly suggest that epigenetic inactivation of NALCN may be a predictor of metastasis in NSCLC. Our results should be validated in further studies based on a larger patient cohort to further investigate whether DNA methylation of the NALCN promoter could serve as a potential prognostic DNA methylation biomarker and predictor of metastasis in NSCLC.

Diversity of the Circulating Tumor Markers: Perspectives of a Multimodal Liquid Biopsy.

Over the past decade, liquid biopsy(LB) has become a routine diagnostic test essential for the treatment of malignant tumors of various localizations. Its capabilities include early diagnosis, molecular genotyping, prognosis, prediction, and monitoring of tumor response. Typically, liquid biopsy involves the extraction of a single type of tumor-derived molecules or cellular elements from blood and subsequent molecular analysis. These elements may include circulating tumor DNA(ctDNA), circulating tumor cells(CTCs), circulating tumor RNA(ctRNA), or contents of extracellular vesicles (exosomes). Despite the technical sophistication of molecular analysis methods for circulating biomarkers, this diagnostic approach has limited relevance. In a significant proportion of cancer patients (ranging from 10 to 50%, depending on the tumor type), none of these analytes can be detected and analyzed, even in the presence of large, progressing neoplastic foci in the body. It seems reasonable to suggest that heterogeneous fractions of the circulating tumor-specific biomarkers complement each other, thus simultaneous analysis of several fractions will not only increase sensitivity of the method but also more accurately characterize and predict the clinical situation. This review examines the possibilities and advantages of applying a combined multiparametric approach to liquid biopsy, which involves testing multiple circulating analytes in a single blood sample.

Integrated Imaging Probe and Bispecific Antibody Development Enables In Vivo Targeting of Glypican-3-Expressing Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a proteoglycan with high sensitivity and specificity for hepatocellular carcinoma (HCC). We describe the integrated development and validation of a GPC3-targeting optical imaging probe and T cell-redirecting antibody (TRAB) as a theranostic strategy for the detection and treatment of HCC. A novel TRAB targeting GPC3 on HCC tumor cells and the CD3 T-cell receptor as well as a distinct GPC3-specific optical imaging probe were developed from a short peptide. The efficacy of GPC3/CD3 TRAB was evaluated in vitro using IFNγ release and calcein-AM assays. Patient-derived xenografts were used to assess the in vivo efficacy of GPC3/CD3 TRAB and the GPC3 imaging probe for the detection of GPC3+ HCC. GPC3/CD3 TRAB caused a dose-dependent escalation in IFNγ release from inactive peripheral blood T cells (P = 0.001) and higher tumor-cell lysis (P = 0.01) compared with controls in vitro. Intratumorally injected GPC3/CD3 TRAB resulted in significant prolongation of tumor doubling time in the GPC3+ tumors, with an associated reduction of tumor fluorescent signal from the HiLyte 488-conjugated GPC3-specific peptide on optical imaging. These data demonstrate that HCC cell targeting using a GPC3/CD3 TRAB derived from a small peptide enabled effective T-cell activation and induction of a cytotoxic response toward GPC3+ HCC tumor cells both in vitro and in vivo. GPC3-specific optical imaging enabled the detection of the GPC3+ HCC cells and noninvasive monitoring of tumor response to adoptive immunotherapy. The integrated development of a targeted therapeutic and molecular imaging probe provides a promising paradigm for the development of cancer theranostics.

Dedicated Breast CT: Getting Ready for Prime Time.

Dedicated breast CT is an imaging modality that provides true 3D imaging of the breast with many advantages over current conventional breast imaging modalities. The addition of intravascular contrast increases the sensitivity of breast CT substantially. As such, there are immediate potential applications in the clinical workflow. These include using breast CT to replace much of the traditional diagnostic workup when faced with indeterminate breast lesions. Contrast-enhanced breast CT may be appropriate as a supplemental screening tool for women at high risk of breast cancer, similar to breast MRI. In addition, emerging studies are demonstrating the utility of breast CT in neoadjuvant chemotherapy tumor response monitoring as well as planning for surgical treatment options. While short exam times and fully 3D imaging in a noncompressed position are advantages of this modality, limited coverage of chest wall/axilla due to prone positioning and use of ionizing radiation are drawbacks. To date, several studies have reported on the performance characteristics of this promising modality.

Contrast-enhanced photon-counting micro-CT of tumor xenograft models

Objective. Photon-counting micro-computed tomography (micro-CT) is a major advance in small animal preclinical imaging. Small molecule- and nanoparticle-based contrast agents have been widely used to enable the differentiation of liver tumors from surrounding tissues using photon-counting micro-CT. However, there is a notable gap in the application of these market-available agents to the imaging of breast and ovarian tumors using photon-counting micro-CT. Herein, we have used photon-counting micro-CT to determine the effectiveness of these contrast agents in differentiating ovarian and breast tumor xenografts in live, intact mice. Approach. Nude mice carrying different types of breast and ovarian tumor xenografts (AU565, MDA-MB-231 and SKOV-3 human cancer cells) were injected with ISOVUE-370 (a small molecule-based agent) or Exitron Nano 12000 (a nanoparticle-based agent) and subjected to photon-counting micro-CT. To improve tumor visualization using photon-counting micro-CT, we developed a novel color visualization method, which changes color tones to highlight contrast media distribution, offering a robust alternative to traditional material decomposition methods with less computational demand. Main results. Our in vivo experiments confirm the effectiveness of this color visualization approach, showing distinct enhancement characteristics for each contrast agent. Qualitative and quantitative analyses suggest that Exitron Nano 12000 provides superior vasculature enhancement and better quantitative consistency across scans, while ISOVUE-370 delivers a more comprehensive tumor enhancement but with significant variance between scans due to its short blood half-time. Further, a paired t-test on mean and standard deviation values within tumor volumes showed significant differences between the AU565 and SKOV-3 tumor models with the nanoparticle-based contrast agent (p-values < 0.02), attributable to their distinct vascularity, as confirmed by immunohistochemical analysis. Significance. These findings underscore the utility of photon-counting micro-CT in non-invasively assessing the morphology and anatomy of different tumor xenografts, which is crucial for tumor characterization and longitudinal monitoring of tumor progression and response to treatments.

Role of [18F]FDG-PET/CT in Evaluation of Tumor Response to Chemoradiation Therapy for Advanced Colorectal Cancer

Objectives: The objective is to evaluate the efficacy of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) computed tomography (CT) in the evaluation of tumor response to preoperative/palliative chemoradiotherapy (CRT) for advanced colorectal cancer; including metastatic cancer at primary presentation and recurrent cancers with local and/or distant metastasis. Materials and Methods: Fifty patients with advanced rectal cancer underwent two point imaging with 18 FDG PET-CT before and after 3 weeks of completion of preoperative/palliative CRT in between 2016 and 2022. Patients with locally recurrent cancer also underwent radical surgery. The assessment consisted of the evaluation of the following metabolic PET parameters: Maximum standardized uptake value (SUVmax), SUVratio, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Response was assessed among the followed patients using RECIST 1.1 criteria. Observations and Results: There was a significant decline in the mean post therapy SUVmax and SUVratio as compared to baseline (P = 0.0001). Twenty-six out of 50 (52%) patients were classified as responders. A significant decrease in all parameters (SUVmax, SUVratio, TLG, and MTV) from baseline was observed in responders of the study when comparing with nonresponders (P &lt; 0.05). Besides SUVmax and SUVratio, the mean posttherapy TLG was significantly reduced in responders than nonresponders (P = 0.0065). Conclusion: PET-CT is a useful combined anatomic and functional imaging modality in monitoring tumor response to preoperative/palliative CRT in advanced rectal cancer, whether primary or recurrent, including metastatic cancers at presentation. Posttherapy SUV and TLG in particular are significantly associated with treatment response.

IMG-02. USING MRI RADIOMICS AND MACHINE LEARNING TO PREDICT OVERALL SURVIVAL FOR PEDIATRIC DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Pediatric diffuse midline gliomas (DMG) are aggressive central nervous system tumors with a median overall survival (OS) of less than one year from diagnosis. There is no curative therapy for DMG, and radiation therapy (RT) is the standard treatment. Magnetic resonance imaging (MRI) is the common noninvasive tool for DMG diagnosis and monitoring of tumor response to therapy. We developed an automatic pipeline to segment subregions of DMG and select MRI features that predict patient OS. METHODS We acquired diagnostic and post-RT multisequence MRI (T1, T1ce, T2, T2 FLAIR) and manual segmentations of pediatric DMG patients from two centers: 53 from Children’s National Hospital (internal) and 16 from Children’s Hospital of Philadelphia (external). We pretrained a deep learning model on a public adult brain tumor dataset, and finetuned the model to segment tumor core (TC) and whole tumor (WT) of DMG. We used PyRadiomics and sequential feature selection to select discriminative features based on the segmented volumes. Two machine learning models were trained on our internal cohort to predict patient 1-year survival from diagnosis. One model used only diagnostic tumor features and the other used both diagnostic and post-RT features. RESULTS For segmentation, Dice score (mean [median]±SD) was 0.91 (0.94)±0.12 and 0.74 (0.83)±0.32 for TC, and 0.88 (0.91)±0.07 and 0.86 (0.89)±0.06 for WT for internal and external cohorts, respectively. For OS prediction, accuracy was 77% and 81% at time of diagnosis, and 85% and 78% post-RT for internal and external cohorts, respectively. Heterogeneous WT intensity in baseline T2 FLAIR and smaller post-RT TC/WT volume ratio indicate longer OS. CONCLUSIONS Our method accurately predict OS for DMG patients and can be extended to other rare pediatric brain tumors. With automated and standardized analysis, early prognostication of OS can guide patient risk stratification and clinical decisions.

Role of dynamic ctDNA monitoring in cervical and anal epidermoid carcinomas under curative chemoradiation.

3141 Background: Cervical and anal canal squamous cell carcinoma (SSC) are a significant health problem in underdevelopment countries. Definitive chemoradiation (CRT) is the standard-of-care (SOC) approach for curative treatment in locally advanced disease. Due to the common substantial local inflammation during CRT, the conventional clinical image cannot identify non-responders early. In this scenario, the evaluation of circulating tumor DNA (ctDNA) is a promising tool for real-time tumor response monitoring. Methods: We performed a prospective multicentric cohort study of patients treated between 2020 and 2023 in tertiary oncologic centers in Brazil to evaluate the role of ctDNA dynamic monitoring in epidermoid cervical (CC) and anal cancer (AC) T1-4, N0-1, M0 by AJCC 8th edition and candidates to complete curative CRT. The cDNA was assessed by Signatera test at D1, D29, immediately post-treatment, 8 weeks (w) post-CRT, 24w post-CRT, every 6 months (m) in the first 1-2 years(y), and yearly at 3-5y of follow-up (FUP). The primary endpoint was to estimate the correlation of ctDNA with a tumoral response assessed by conventional routine image and clinical evaluation at 8w. Secondary endpoints included a correlation between ctDNA results at different time points with progression-free survival (PFS) and overall survival (OS). The predictive potential of the biomarker was evaluated using receiving operating characteristic (ROC) curve analysis. Results: We included 33 patients, and 27 were evaluable with ctDNA, with a median FUP of 10m. The majority were female (n=23, 85.1%), and 3 (11%) were HIV-positive(+). Most patients presented with positive nodes and stage III disease (n=18, 66.6%). In the AC group (n=15), the majority received CRT with capecitabine and cisplatin (n=12, 55.5%); in the CC group (n=12), all pts received cisplatin. All pts tested expressed ctDNA+ before treatment. At 8w, images had a sensitivity of 42.8% and specificity of 100% for disease progression (area under the curve [AUC] 0.714), while ctDNA yielded a sensitivity of 85.7% and specificity of 89.4% (AUC 0.875). The ctDNA+ immediately after CRT ended (32%) was consistent with ctDNA+ at 8w (30.7%) and 24w (30%). Pts with ctDNA+ immediately post CRT have a higher risk of disease progression with PFS of 8.2m in ctDNA+ and not reached in ctDNA- group (HR:17.5; IC95%:1.9-157.3; p=0.01). Data are immature for OS analysis. Conclusions: CtDNA immediately post-CRT has a high predictive value in patients with anal and cervical tumors in early access CRT non-responders, who are at a high risk of disease progression. This biomarker should be considered for tailoring strategies of treatment escalation in this population.

Deciphering Tumor Response: The Role of Fluoro-18-d-Glucose Uptake in Evaluating Targeted Therapies with Tyrosine Kinase Inhibitors.

Background/Objectives: The inhibitory effects of tyrosine kinase inhibitors (TKIs) on glucose uptake through their binding to human glucose transporter-1 (GLUT-1) have been well documented. Thus, our research aimed to explore the potential impact of various TKIs of GLUT-1 on the standard [18F]FDG-PET monitoring of tumor response in patients. Methods: To achieve this, we conducted an analysis on three patients who were undergoing treatment with different TKIs and harbored actionable alterations. Alongside the assessment of FDG data (including SUVmax, total lesion glycolysis (TLG), and metabolic tumor volume (MTV)), we also examined the changes in tumor sizes through follow-up [18F]FDG-PET/CT imaging. Notably, our patients harbored alterations in BRAFV600, RET, and c-KIT and exhibited positive responses to the targeted treatment. Results: Our analysis revealed that FDG data derived from SUVmax, TLG, and MTV offered quantifiable outcomes that were consistent with the measurements of tumor size. Conclusions: These findings lend support to the notion that the inhibition of GLUT-1, as a consequence of treatment efficacy, could be indirectly gauged through [18F] FDG-PET/CT imaging in cancer patients undergoing TKI therapy.

Predicting therapeutic response to neoadjuvant immunotherapy based on an integration model in resectable stage IIIA (N2) non-small cell lung cancer

ObjectiveAccurately predicting response during neoadjuvant chemoimmunotherapy for resectable non-small cell lung cancer (NSCLC) remains clinically challenging. In this study, we investigate the effectiveness of blood-based tumor mutational burden (bTMB) and a deep learning (DL) model in predicting major pathologic response (MPR) and survival from a phase II trial. MethodsBlood samples were prospectively collected from 45 stage IIIA (N2) NSCLC patients undergoing neoadjuvant chemoimmunotherapy. An integrated model, combining the CT-based DL score, bTMB, and clinical factors, was developed to predict tumor response to neoadjuvant chemoimmunotherapy. ResultsAt baseline, bTMB were detected in 77.8% (35 of 45) of patients. Baseline bTMB ≥11 Muts/Mb was associated with significantly higher MPR rates (77.8% vs. 38.5%, p = 0.042), and longer disease-free survival (DFS, p = 0.043), but not overall survival (p = 0.131), compared to bTMB < 11 Muts/Mb in 35 patients with bTMB available. The developed DL model achieved an area under the curve (AUC) of 0.703 in all patients. Importantly, the predictive performance of the integrated model improved to an AUC of 0.820 when combining the DL score with bTMB and clinical factors. Baseline circulating tumor DNA (ctDNA) status was not associated with pathological response and survival. Compared to ctDNA residual, ctDNA clearance before surgery was associated with significantly higher MPR rates (88.2% vs. 11.1%, p < 0.001) and improved DFS (p = 0.010). ConclusionsThe integrated model shows promise as a predictor of tumor response to neoadjuvant chemoimmunotherapy. Serial ctDNA dynamics provide a reliable tool for monitoring tumor response.

Noninvasively Deciphering the Immunosuppressive Tumor Microenvironment Using Galectin-1 PET to Inform Immunotherapy Responses.

Immune checkpoint blockade (ICB) has achieved groundbreaking results in clinical cancer therapy; however, only a subset of patients experience durable benefits. The aim of this study was to explore strategies for predicting tumor responses to optimize the intervention approach using ICB therapy. Methods: We used a bilateral mouse model for proteomics analysis to identify new imaging biomarkers for tumor responses to ICB therapy. A PET radiotracer was synthesized by radiolabeling the identified biomarker-targeting antibody with 124I. The radiotracer was then tested for PET prediction of tumor responses to ICB therapy. Results: We identified galectin-1 (Gal-1), a member of the carbohydrate-binding lectin family, as a potential negative biomarker for ICB efficacy. We established that Gal-1 inhibition promotes a sensitive immune phenotype within the tumor microenvironment (TME) for ICB therapy. To assess the pre-ICB treatment status of the TME, a Gal-1-targeted PET radiotracer, 124I-αGal-1, was developed. PET imaging with 124I-αGal-1 showed the pretreatment immunosuppressive status of the TME before the initiation of therapy, thus enabling the prediction of ICB resistance in advance. Moreover, the use of hydrogel scaffolds loaded with a Gal-1 inhibitor, thiodigalactoside, demonstrated that a single dose of thiodigalactoside-hydrogel significantly potentiated ICB and adoptive cell transfer immunotherapies by remodeling the immunosuppressive TME. Conclusion: Our study underscores the potential of Gal-1-targeted PET imaging as a valuable strategy for early-stage monitoring of tumor responses to ICB therapy. Additionally, Gal-1 inhibition effectively counteracts the immunosuppressive TME, resulting in enhanced immunotherapy efficacy.

Towards tumour hypoxia imaging: Incorporating relative oxygen extraction fraction mapping of prostate with multi-parametric quantitative MRI on a 1.5T MR-linac.

Hypoxia plays a central role in tumour radioresistance. Reliable tumour hypoxia imaging would allow the monitoring of tumour response and a more personalized adaptation of radiotherapy planning. Here, we showed a proof of concept of the feasibility and repeatability of relative oxygen extraction fraction (rOEF) mapping of prostate using multi-parametric quantitative MRI (qMRI) achieved for the first time on a 1.5T MR-linac. T2, T2* relaxation times maps, and intra-voxel incoherent motion (IVIM) parametric maps mapping were computed on a 29 years old healthy volunteer. R2' and rOEF maps werecalculated based on a multi-parametric model. Long-term repeatability and repeatability coefficient (RC) were determined for each parameter according to QIBA recommendations. Mean values for the entire healthy prostate were 0.99 ± 0.14 × 10-3 mm/s2, 81 ± 2.1 × 10-3 mm/s2, 21.6 ± 3.6%, 92.7 ± 19.7 ms and 62.4 ± 17.3 ms for Dslow, Dfast, f, T2 and T2*, respectively. R2' and rOEF in the prostate were 6.1 ± 3.4 s-1 and 18.2 ± 10.1% respectively. The RC of rOEF was 4.43%. Long-term repeatability of quantitative parameters based on a test-retest ranged from 2 to 18%. qMRI parameters are measurable and repeatable on 1.5T MR LINAC. From T2, T2* and IVIM parameters maps, we were able to obtain a rOEF mapping of the prostate.These results are the first step to a non-invasive imaging of tumour hypoxia during radiotherapy leading to a biological image-guided adaptive radiotherapy.

Abstract A036: Quantitative molecular imaging of pancreatic tumor fibrosis for evaluation of response to neoadjuvant therapy: Pre-clinical and first-in-human application of collagen PET/MRI

Abstract Purpose: One of the major challenges in the management of pancreatic ductal adenocarcinoma (PDAC) is the lack of a reliable imaging tool to monitor tumor response to treatment. A high degree of fibrosis, mainly collagen type I, has been recognized as the hallmark of PDAC, which further increases in response to neoadjuvant chemoradiotherapy (CRT). This study introduces an image-guided paradigm for monitoring treatment response using the collagen type I specific PET imaging probe, 68Ga-CBP8 in mouse models and patients with PDAC. Methods: Subcutaneous mouse models of human PDAC were generated using FOLFIRNOX-sensitive (PANC1 and PDAC6) and FOLFIRINOX-resistant (SU8686) PDAC cells in nude mice (n=88). Mice were randomized into 2 groups of treatment with FOLFIRINOX or vehicle i.v. twice a week. Animals underwent PET/MRI with 68Ga-CBP8 (3.7-11.1 MBq) prior to, and at 7- and 15-days post-treatment (n=4-8/group). To assess the specificity of the probe, additional PANC1 tumor-bearing mice (n=4) were imaged with 68Ga-CBP8, and a collagen non-binding probe, 68Ga-CNBP (3.7-11 MBq). PET values were correlated with tumor growth over 21 days and quantification of fibrosis by Picrosirius red and IHC for collagen I, α-SMA, FAP-α, collagen rtPCR, and hydroxyproline assay. 8 patients (49-65 yrs.) with newly diagnosed PDAC underwent 68Ga-CBP8 PET (122-281 MBq) and standard MRI. Thus far, 3/8 subjects have undergone repeat PET/MRI after completion of neoadjuvant CRT. The pre-and post-CRT PET parameters for tumor and pancreas were compared and correlated with tumor size change, histology, and spatial analyses for PDAC and fibroblasts. Results: PET/MRI of PANC1 tumors showed significantly higher 68Ga-CBP8 uptake compared to control 68Ga-CNBP at each time point (p &amp;lt; 0.05), demonstrating the specificity of the probe in targeting collagen. PET values significantly increased over time in FOLFIRINOX-sensitive tumors (PANC1 %ID/cc: 0.8 ± 0.2 vs. 1.1 ± 0.3 vs. 1.9 ±0.2, p &amp;lt; 0.05 in day-0 vs. day-7 vs. day-15, respectively, p = 0.0005), whereas no significant change was seen in the probe uptake in the vehicle or resistant tumors (p &amp;gt; 0.05). There was a significant tumor growth reduction in responders compared to vehicle and resistant tumors. Histology showed a continuous increase of collagen in responding tumors (% collagen proportion area (CPA) in PANC1: 5.2 ± 3.8 vs. 16.2 ± 8.7 vs. 46.9 ± 6.0, p &amp;lt; 0.05, at Day-0 vs. Day-7 vs. Day-15, respectively, p &amp;lt; 0.0001), no change in resistant SU8686 (CPA: 33.0 ± 8.9 vs. 30.0 ± 9.7 vs. 28.1 ± 7.2, at Day-0 vs. Day-7 vs. Day-15, respectively, p = 0.66) and unchanged minimal collagen in pancreas. PET/MRI of patients showed a significantly higher SUVmean in the tumor compared to pancreas (2.4 ± 0.37 vs. 1.9 ± 0.31, p = 0.01), and a significantly higher uptake in the treated compared to untreated tumors (SUVmean: 2.8 ± 0.33 vs. 1.9 ± 0.56, p = 0.04). Response to treatment was confirmed on histology. Conclusion: Collagen PET could be used as a reliable non-invasive tool for monitoring response to neoadjuvant therapy in PDAC. Citation Format: Shadi A. Esfahani, Hua Ma, Shriya Krishna, Sergey Shuvaev, Arvin Hajmirzaian, Iris Zhou, Ciprian Catana, Mozhdeh Sojoodi, Eric Abston, David Ting, Pedram Heidari, Michael Lanuti, Kenneth Tanabe, Motaz Qadan, Carlos Fernandez de-Castillo, Aparna Parikh, Colin Weekes, Theodore Hong, Peter Caravan. Quantitative molecular imaging of pancreatic tumor fibrosis for evaluation of response to neoadjuvant therapy: Pre-clinical and first-in-human application of collagen PET/MRI [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A036.

Preclinical Application of CEST MRI to Detect Early and Regional Tumor Response to Local Brain Tumor Treatment.

Treating glioblastoma and monitoring treatment response non-invasively remain challenging. Here, we developed a robust approach using a drug-loaded liposomal hydrogel that is mechanically compatible with the brain, and, simultaneously, we successfully monitored early tumor response using Chemical Exchange Saturation Transfer (CEST) MRI. This CEST-detectable liposomal hydrogel was optimized based on a sustainable drug release and a soft hydrogel for the brain tumor, which is unfavorable for tumor cell proliferation. After injecting the hydrogel next to the tumor, three distinctive CEST contrasts enabled the monitoring of tumor response and drug release longitudinally at 3T. As a result, a continuous tumor volume decrease was observed in the treatment group along with a significant decrease in CEST contrasts relating to the tumor response at 3.5 ppm (Amide Proton Transfer; APT) and at -3.5 ppm (relayed Nuclear Overhauser Effect; rNOE) when compared to the control group (p < 0.05). Interestingly, the molecular change at 3.5 ppm on day 3 (p < 0.05) was found to be prior to the significant decrease in tumor volume on day 5. An APT signal also showed a strong correlation with the number of proliferating cells in the tumors. This demonstrated that APT detected a distinctive decrease in mobile proteins and peptides in tumors before the change in tumor morphology. Moreover, the APT signal showed a regional response to the treatment, associated with proliferating and apoptotic cells, which allowed an in-depth evaluation and prediction of the tumor treatment response. This newly developed liposomal hydrogel allows image-guided brain tumor treatment to address clinical needs using CEST MRI.

IRGD-mediated liposomal nanoplatforms for improving hepatocellular carcinoma targeted combination immunotherapy and monitoring tumor response via IVIM-MRI.

The combination therapy of targeted treatments and immune checkpoint blockade (ICB) holds great promise for hepatocellular carcinoma (HCC) treatment. However, challenges such as immunogenicity, off-target toxicity of ICB antibodies, low drug co-delivery efficiency, and lack of effective biomarkers to monitor treatment response limit the efficacy of existing targeted immunotherapies. Herein, we synthesized iRGD-modified pH-sensitive liposomal nanoparticles co-encapsulating lenvatinib (Len) and the small molecule PD-1/PD-L1 inhibitor BMS-202 (iRGD-lip@Len/BMS-202) to address issues related to inadequate tumor enrichment and distinct pharmacokinetics of these drugs. Furthermore, intravoxel incoherent motion-magnetic resonance imaging (IVIM-MRI), which is calculated using a biexponential model, can simultaneously reflect both the diffusion of water molecules within the tissue and the microcirculatory perfusion of capillaries. Consequently, we further assessed the feasibility of using IVIM-MRI to monitor the cancer treatment response in nanodrug therapy. These results demonstrated that the iRGD-targeted liposomal nanodrug effectively accumulated in tumors and released in acidic microenvironments. The sustained release of Len facilitated tumor vascular normalization, decreased the presence of Tregs and MDSCs and activated the IFN-γ signaling pathway. This led to increased PD-L1 expression in tumor cells, enhancing the sensitivity of BMS-202. Consequently, there was a synergistic amplification of antitumor immune therapy, resulting in the shrinkage of subcutaneous and orthotopic HCC and inhibition of lung metastasis. Furthermore, IVIM-MRI technology facilitated the non-invasive monitoring of the tumor microenvironment (TME), revealing critical therapeutic response indicators such as the normalization of tumor blood vessels and the degree of hypoxia. Collectively, the combination of Food and Drug Administration (FDA)-approved drugs with iRGD-modified liposomes presents a promising strategy for HCC treatment. Simultaneously, IVIM-MRI provides a non-invasive method to accurately predict the response to this nanodrug.

Tumor volume features predict survival outcomes for patients diagnosed with diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood central nervous system tumor. Diagnosis and monitoring of tumor response to therapy is based on magnetic resonance imaging (MRI). MRI-based analyses of tumor volume and appearance may aid in the prediction of patient overall survival (OS). Contrast-enhanced T1- and FLAIR/T2-weighted MR images were retrospectively collected from children with classical DIPG diagnosed by imaging (n = 43 patients). MRI features were evaluated at diagnosis (n = 43 patients) and post-radiation (n = 40 patients) to determine OS outcome predictors. Features included 3D tumor volume (Twv), contrast-enhancing tumor core volume (Tc), Tc relative to Twv (TC/Twv), and Twv relative to whole brain volume. Support vector machine (SVM) learning was used to identify feature combinations that predicted OS outcome (defined as OS shorter or longer than 12 months from diagnosis). Features associated with poor OS outcome included the presence of contrast-enhancing tumor at diagnosis, >15% Tc/Twv post-radiation therapy (RT), and >20% ∆Tc/Twv post-RT. Consistently, SVM learning identified Tc/Twv at diagnosis (prediction accuracy of 74%) and ∆Tc/Twv at <2 months post-RT (accuracy = 75%) as primary features of poor survival. This study demonstrates that tumor imaging features at diagnosis and within 4 months of RT can predict differential OS outcomes in DIPG. These findings provide a framework for incorporating tumor volume-based predictive analyses into the clinical setting, with the potential for treatment customization based on tumor risk characteristics and future applications of machine-learning-based analysis.

Collagen type I PET/MRI enables evaluation of treatment response in pancreatic cancer in pre-clinical and first-in-human translational studies.

Pancreatic ductal adenocarcinoma (PDAC) is an invasive and rapidly progressive malignancy. A major challenge in patient management is the lack of a reliable imaging tool to monitor tumor response to treatment. Tumor-associated fibrosis characterized by high type I collagen is a hallmark of PDAC, and fibrosis further increases in response to neoadjuvant chemoradiotherapy (CRT). We hypothesized that molecular positron emission tomography (PET) using a type I collagen-specific imaging probe, 68Ga-CBP8 can detect and measure changes in tumor fibrosis in response to standard treatment in mouse models and patients with PDAC. Methods: We evaluated the specificity of 68Ga-CBP8 PET to tumor collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in nude mouse models of human PDAC including FOLFIRNOX-sensitive (PANC-1 and PDAC6) and FOLFIRINOX-resistant (SU.86.86). Next, we demonstrated the specificity and sensitivity of 68Ga-CBP8 to the deposited collagen in resected human PDAC and pancreas tissues. Eight male participant (49-65 y) with newly diagnosed PDAC underwent dynamic 68Ga-CBP8 PET/MRI, and five underwent follow up 68Ga-CBP8 PET/MRI after completing standard CRT. PET parameters were correlated with tumor collagen content and markers of response on histology. Results: 68Ga-CBP8 showed specific binding to PDAC compared to non-binding 68Ga-CNBP probe in two mouse models of PDAC using PET imaging and to resected human PDAC using autoradiography (P < 0.05 for all comparisons). 68Ga-CBP8 PET showed 2-fold higher tumor signal in mouse models following FOLFIRINOX treatment in PANC-1 and PDAC6 models (P < 0.01), but no significant increase after treatment in FOLFIRINOX resistant SU.86.86 model. 68Ga-CBP8 binding to resected human PDAC was significantly higher (P < 0.0001) in treated versus untreated tissue. PET/MRI of PDAC patients prior to CRT showed significantly higher 68Ga-CBP8 uptake in tumor compared to pancreas (SUVmean: 2.35 ± 0.36 vs. 1.99 ± 0.25, P = 0.036, n = 8). PET tumor values significantly increased following CRT compared to untreated tumors (SUVmean: 2.83 ± 0.30 vs. 2.25 ± 0.41, P = 0.01, n = 5). Collagen deposition significantly increased in response to CRT (59 ± 9% vs. 30 ± 9%, P=0.0005 in treated vs. untreated tumors). Tumor and pancreas collagen content showed a positive direct correlation with SUVmean (R2 = 0.54, P = 0.0007). Conclusions: This study demonstrates the specificity of 68Ga-CBP8 PET to tumor type I collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in PDAC. The results highlight the potential use of collagen PET as a non-invasive tool for monitoring response to treatment in patients with PDAC.

Dynamic Monitoring of Circulating Tumor DNA in Patients With Metastatic Colorectal Cancer.

Plasma circulating tumor DNA (ctDNA) is a valuable resource for tumor characterization and for monitoring of residual disease during treatment; however, it is not yet introduced in metastatic colorectal cancer (mCRC) routine clinical practice. In this retrospective exploratory study, we evaluated the role of ctDNA in patients with mCRC treated with chemotherapy plus bevacizumab. Fifty-three patients were characterized for RAS and BRAF status on tumor tissue before the start of treatment. Plasma was collected at baseline, at first clinical evaluation, and at disease progression. ctDNA analysis was performed using Oncomine Colon cfDNA Assay on the Ion S5 XL instrument. At baseline, from a plasma sample, RAS, BRAF, or PIK3CA mutations were detected in 44 patients. A high correspondence was observed between ctDNA and tumor tissue mutations (KRAS 100%, NRAS 97.9%, BRAF 97.9%, PIK3CA 90%). Low baseline variant allele frequency (VAF) was found to be associated with longer median progression-free survival (PFS) compared with those with high VAF (15.9 v 12.2 months, P = .02). A higher PFS {12.29 months (95% CI, 9.03 to 17.9) v 8.15 months (95% CI, 2.76 to not available [NA]), P = .04} and overall survival (34.1 months [95% CI, 21.68 to NA] v 11.1 months [95% CI, 3.71 to NA], P = .003) were observed in patients with large decline in VAF at first evaluation. ctDNA analysis is useful for molecular characterization and tumor response monitoring in patients with mCRC. Quantitative variations of released ctDNA are associated with clinical outcomes.

RNA reporter based CRISPR/Cas12a biosensing platform for sensitive detection of circulating tumor DNA.

CRISPR/Cas biotechnology provides an exceptional platform for biosensor development. To date, the reported CRISPR/Cas biosensing systems have shown extraordinary performance for nucleic acids, small molecules, small proteins and microorganism detection. The CRISPR/Cas12a biosensing system, as a typical example, has been well established and applied for both nucleic acids and non-nucleic acids target detection. However, all established CRISPR/Cas12a biosensing systems are based on DNA reporters, which potentially limits further application.In this study, we established an RNA reporter based CRISPR/Cas12a biosensing system. A basic biosensing system was evaluated, and the limit of detection was found to be 1 nM. Afterwards, we optimized this biosensing system using both temperature and chemical enhancers. The final optimal biosensing system (with DTT & 37°C) shows fluorescence signal increased by a factor of ~10 compared with the basic system. The optimal biosensing system was further applied for the detection of circulating tumor DNA (ctDNA), which shows over 4 orders of magnitude detection range from 1pM to 25 nM, with the limit of detection of 1pM. This RNA reporter based CRISPR/Cas12a biosensing system provides an effective platform for nucleic acids quantification.Clinical Relevance- This research provides a novel approach for ctDNA diagnostics, which is an attractive biomarker for noninvasive monitoring of tumor growth, response, and spread.

Retrospective clinical analysis of circulating tumor DNA (ctDNA)–based molecular response (MR) and baseline blood-based tumor mutational burden (bTMB) for monitoring response in phase 3 trial of bintrafusp alfa vs. pembrolizumab treatment of non-small cell lung cancer (NSCLC).

9098 Background: The clinical utility of ctDNA-based approaches, such as bTMB and MR, to monitor and predict response during chemo-immunotherapy was evaluated using longitudinal cohort samples from a Phase 3 study of bintrafusp alfa versus pembrolizumab as a first-line treatment in patients with PD-L1 expressing advanced NSCLC. Methods: ctDNA was collected at baseline and day 42 after treatment with bintrafusp alfa (Q2W, 1200mg) or pembrolizumab (Q3W, 200mg). The GuardantOMNI liquid biopsy (LBx) assay was used to detect somatic alterations in 497 genes and generate bTMB from baseline, and MR scores from baseline and day 42 (n = 424 samples). bTMB and somatic alterations influencing response were explored. MR scores were calculated using the validated Guardant Response algorithm. Associations between ctDNA metrics and PFS were assessed. Results: 418/424 samples passed sequencing QC; of these, somatic mutations were detected in 236/237 baseline samples and 177/181 day 42 samples. Of 212 NSCLC patients with eligible bTMB results at baseline, 104 (49.1%) patients received bintrafusp alpha and 108 (50.9%) received pembrolizumab. bTMB high (bTMB-H) is defined as &gt;20mut/MB. In the bintrafusp alpha arm, PFS was significantly longer in patients with bTMB-H (median 8.3 months vs 2.7 months, p = 0.00086). In the pembrolizumab arm, PFS was also longer in patients with bTMB-H (median 5.65 months vs 5.5 months, p = 0.0898). Of 132 NSCLC patients with eligible MR results, 67 (50.8%) patients received bintrafusp alpha and 65 (49.2%) patients received pembrolizumab. In the bintrafusp alpha arm, molecular responders had significantly longer PFS (median 7.9 months vs 4.2 months, p = 0.00042). In the pembrolizumab arm, molecular responders also had significantly longer PFS (median 8.95 months vs 4.1 months, p = 0.00054). Conclusions: ctDNA analysis from plasma samples supported MR assessment in patients treated with ICI, indicating its utility as an adjunct to RECIST in monitoring of tumor response. Blood-based TMB-high was associated with immunotherapy treatment benefit. Analysis of b-TMB and MR allowed identification of patients with improved PFS in both treatment arms. Investigation of treatment specific TMB cut off selection and potential clinical utility of defined somatic mutations is ongoing. Clinical trial information: NCT03631706 .