Monitoring Cancer Treatment Response Research Articles

Impact of panel size on minimum residual disease (MRD) assay performance.

e15022 Background: Sequencing circulating tumor DNA is a promising method for monitoring cancer treatment response and detecting recurrence, but sensitivity at the low tumor fractions typical of early-stage, post-treatment, and early recurrent tumors is limited by the small number of variants targeted in commercially available assays. Methods: We developed a tumor-informed minimum residual disease (MRD) assay using tumor-normal whole-genome sequencing (WGS) followed by interrogation of select somatic variants in cell-free DNA (cfDNA). We describe theoretical limits on panel designability as a function of tumor purity and sequencing depth using simulations and a discovery cohort of 31 patient samples. cfDNA assay performance was tested by creating serial dilutions of patient plasma before somatic enrichment and sequencing. Simulations and in-silico downsampling of target sites were used to test sensitivity as a function of panel size. A bioinformatics pipeline incorporating somatic calling, copy number inference, error assessment, and tumor-fraction estimation was developed to analyze results. Results: Across a range of tissues, tumor-normal WGS identified from hundreds to tens of thousands of somatic variants to inform an MRD capture panel. 97% of samples tested returned at least 300 high-confidence somatic variants, and targeted sequencing demonstrated over 95% positive predictive value in somatic variant calls. In cfDNA dilution experiments, we observed > 99% sensitivity at a tumor fraction of 0.01%. Expanding the panel size from 16 to 300 target variants increased sensitivity at 0.005% tumor fraction from approximately 20% to > 95%. Our tumor fraction model returned accurate quantification down to the parts per hundred thousand (0.001%) range, even when only a single tumor molecule could be observed at each target site. Conclusions: Increasing the number of targeted variants in an MRD assay increases sensitivity at low tumor fractions, and WGS-driven panel design ensures a sufficient number of interrogated variants while minimizing the failure rate due to panel designability. High sensitivity MRD has the potential to enable earlier recurrence detection while also giving researchers and clinicians new tools for monitoring patient treatment responses.

Abstract 3099: Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients

Abstract Purpose Circulating tumor DNA (ctDNA) can potentially be used to monitor cancer treatment response. In this study, we developed and used a sensitive and selective ultra-deep next-generation sequencing (NGS) approach for detection of ctDNA in lung cancer patients. Method Most studies use fixed gene panels for detection of ctDNA by NGS. Deep sequencing of large target regions is expensive and generates a myriad of variants, which are difficult to interpret. For the purpose of using ctDNA for monitoring, we try to overcome these challenges by sequencing only selected regions where mutations were previously found in the tumor. This method is more sensitive and cost-efficient. In this study, we included about 80 lung cancer patients. Tumor DNA was sequenced with a large gene panel and somatic mutations were identified in each tumor. We then defined the target region according to the tumor mutations and selected primers for constructing NGS libraries. Since the coverage region is small it enables ultra-deep sequencing of several samples simultaneously. We evaluated the lower limit of detection for this method by sequencing a series of artificially constructed samples with known mutant allele frequencies (MAFs). DNA from peripheral blood leucocytes was sequenced in parallel with plasma DNA to exclude variants from clonal hematopoiesis. Preliminary results Using 40 ng as input DNA, which contains approximately 12,000 haploid genomes, we detected mutations down to 0.02% MAF. This corresponds to about 6 mutated copies per mL plasma. Currently, we are applying this method on patient plasma DNA samples. Conclusion We have developed a sensitive method for ctDNA detection, and we will present the results of the lung cancer patient samples. Citation Format: Anine Larsen Ottestad, Elisabeth F. Emdal, Sissel G. Wahl, Bjørn Henning Grønberg, Hong Yan Dai. Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3099.

Diffusion weighted magnetic resonance imaging (DW-MRI) as a non-invasive, tissue cellularity marker to monitor cancer treatment response

BackgroundDiffusion weighted magnetic resonance imaging (DW-MRI) holds great potential for monitoring treatment response in cancer patients shortly after initiation of radiotherapy. It is hypothesized that a decrease in cellular density of irradiated cancerous tissue will lead to an increase in quantitative apparent diffusion coefficient (ADC) values. DW-MRI can therefore serve as a non-invasive marker of cell death and apoptosis in response to treatment. In the present study, we aimed to investigate the applicability of DW-MRI in preclinical models to monitor radiation-induced treatment response. In addition, we compared DW-MRI with ex vivo measures of cell density, cell death and apoptosis.MethodsDW-MRI was tested in two different syngeneic mouse models, a colorectal cancer (CT26) and a breast cancer (4 T1). ADC values were compared with quantitative determinations of apoptosis and cell death by flow cytometry. Furthermore, ADC-values were also compared to histological measurement of cell density on tumor sections.ResultsWe found a significant correlation between ADC-values and apoptotic state in the CT26 model (P = 0.0031). A strong correlation between the two measurements of ADC-value and apoptotic state was found in both models, which were also present when comparing ADC-values to cell densities.ConclusionsOur findings demonstrate that DW-MRI can be used for non-invasive monitoring of radiation-induced changes in cell state during cancer therapy. ADC values reflect ex vivo cell density and correlates well with apoptotic state, and can hereby be described as a marker for the cell state after therapy and used as a non-invasive response marker.

Water-Soluble and Bright Luminescent Cesium-Lead-Bromide Perovskite Quantum Dot-Polymer Composites for Tumor-Derived Exosome Imaging.

Cesium-lead-halide perovskite quantum dots (PQDs) are a highly promising class of the next-generation optical material for bioimaging applications. Herein, we present a nanocomposite strategy for the design of water-soluble, highly luminescence CsPbBr3 PQD nanocomposites without modifying the crystal symmetry and photoluminescence (PL) property. Water-soluble PQDs are reproducibly synthesized via encapsulating CsPbBr3 PQDs with polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (PS-PEB-PS) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol (PEG-PPG-PEG). In the reported design, the polystyrene triblock polymers strongly interact with the hydrophobic parts of PQDs, and the water-soluble PEG moiety acts as a protection layer to effectively prevent degradation of PQDs in water. The outer shell PEG layer also helps to develop biocompatible PQDs. Reported data indicate that encapsulating CsPbBr3 PQDs with a polymer helps to improve the photoluminescence quantum yield (PLQY) from 83% to 88%, which may be due to a decrease in the surface defects after the effective polymer coating. Experimental data show that the PL intensity from CsPbBr3 PQD nanocomposites remains unchanged even after 30 days of exposure in air. Similarly, reported data indicate that nanocomposites retain their luminescence properties in water for the first 8 days and then decrease slowly to 60% of its initial PL intensity after one month. On the other hand, the PL emission for the PQD without polymer encapsulation is completely quenched within a few hours. Exosomes are a highly promising avenue for accessing tumor type and stage and monitoring cancer treatment response. Reported data reveal that anti-CD63 antibody-attached PQD nanocomposites are capable of tracking triple-negative MDA-MB-231 breast tumor-derived exosomes via binding using anti-CD63 antibody and selective green luminescence imaging using PQD nanocomposites.

Quantitative photoacoustic imaging study of tumours in vivo: Baseline variations in quantitative measurements

Photoacoustic imaging (PAI) provides information on haemoglobin levels and blood oxygenation (sO2). To facilitate assessment of the variability in sO2 and haemoglobin in tumours, for example in response to therapies, the baseline variability of these parameters was evaluated in subcutaneous head and neck tumours in mice, using a PAI system (MSOTinVision-256TF). Tumours of anaesthetized animals (midazolam-fentanyl-medetomidine) were imaged for 75 min, in varying positions, and repeatedly over 6 days. An increasing linear trend for average tumoural haemoglobin and blood sO2 was observed, when imaging over 75 min. There were no significant differences in these temporal trends, when repositioning tumours. A negative correlation was found between the percent decrease in blood sO2 over 6 days and tumour growth rate. This paper shows the potential of PAI to provide baseline data for assessing the significance of intra- and inter-tumoural variations that may eventually have value for predicting and/or monitoring cancer treatment response.

Abstract 57: Detection of cell-free circulating tumor DNA in plasma as a biomarker for treatment response, prognosis, and recurrence in head and neck squamous cell carcinoma

Abstract Objective: Head and neck squamous cell carcinoma (HNSCC) affects approximately 500,000 people per year, with 50,000 affected in the United States. Depending on stage and pathologic features, treatment modalities consist of surgery with or without adjuvant therapy, or primary chemoradiation. However, monitoring with serial examination and imaging including CT or PET-CT has significant limitations with a delay between actual and detectable tumor response. Circulating cell-free tumor DNA (ctDNA) has shown promise as a biomarker for monitoring cancer treatment response, recurrence, and prognosis. We utilize a novel, barcoded next-generation sequencing approach called SimSen-Seq to facilitate ultrasensitive detection of ctDNA, and we are applying it to samples from patients with head and neck squamous cell carcinoma (HNSCC). The purpose of this study is to evaluate the feasibility of using tumor mutations that are detectable in ctDNA to monitor treatment response and recurrence in patients with HNSCC. Methods: Longitudinal blood samples were obtained from patients with Stage I-IV HNSCC, with the initial sample obtained immediately before surgery. Imaging studies and pathology reports were reviewed to determine disease stage. Tumor samples were obtained from surgical specimens, and tumor DNA was sequenced using a targeted panel to identify mutations. SimSen-Seq assays were developed for each patient and used to generate sequencing libraries from cell-free DNA isolated from plasma. Mutations in plasma were identified, quantified, and associations with disease stage and response to therapy were explored. Results: Preliminary results from 5 patients with HNSCC demonstrate detection of ctDNA, and correlation of ctDNA fraction with treatment response and recurrence. Four of these patients had stage IV disease, and ctDNA was detectable immediately preoperatively. One patient demonstrated positive ctDNA 3 weeks postoperatively, with no clinical evidence of residual disease, but subsequently recurred and died soon afterward. Two other patients demonstrated negative ctDNA at 3-week postoperative blood draws, but subsequently demonstrated metastatic recurrence over 6 months later. Conclusions: ctDNA can be detected in the plasma of HNSCC patients, and can be detected prior to clinical evidence of recurrence. Furthermore, positive detection early after surgery may suggest a poor prognosis. Additional samples have been collected, and more work to evaluate detection rates based on stage plus response to therapy and recurrence is ongoing. Citation Format: Stefanie S. Saunders, Matthew Egyud, Anand Devaiah, Scharukh Jalisi, Tony Godfrey. Detection of cell-free circulating tumor DNA in plasma as a biomarker for treatment response, prognosis, and recurrence in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 57.

Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model.

It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo. [89Zr] Keytruda (groups=2; NSG-ctl (control) and hNSG-nblk (non-blocking), n=3-5, 3.2±0.4MBq/15-16μg/200μl) and [64Cu] Keytruda (groups=3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n=4, 7.4±0.4MBq /20-25μg/200μl) were administered in mice. PET-CT scans were performed over 1-144h ([89Zr] Keytruda) and 1-48h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g±SD) of 3.8±0.4 ([89Zr] Keytruda), and 6.4±0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p=0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144h ([89Zr] Keytruda) and 48h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively. Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.

Computer Aided Theragnosis Using Quantitative Ultrasound Spectroscopy and Maximum Mean Discrepancy in Locally Advanced Breast Cancer.

A noninvasive computer-aided-theragnosis (CAT) system was developed for the early therapeutic cancer response assessment in patients with locally advanced breast cancer (LABC) treated with neoadjuvant chemotherapy. The proposed CAT system was based on multi-parametric quantitative ultrasound (QUS) spectroscopic methods in conjunction with advanced machine learning techniques. Specifically, a kernel-based metric named maximum mean discrepancy (MMD), a technique for learning from imbalanced data based on random undersampling, and supervised learning were investigated with response-monitoring data from LABC patients. The CAT system was tested on 56 patients using statistical significance tests and leave-one-subject-out classification techniques. Textural features using state-of-the-art local binary patterns (LBP), and gray-scale intensity features were extracted from the spectral parametric maps in the proposed CAT system. The system indicated significant differences in changes between the responding and non-responding patient populations as well as high accuracy, sensitivity, and specificity in discriminating between the two patient groups early after the start of treatment, i.e., on weeks 1 and 4 of several months of treatment. The proposed CAT system achieved an accuracy of 85%, 87%, and 90% on weeks 1, 4 and 8, respectively. The sensitivity and specificity of developed CAT system for the same times was 85%, 95%, 90% and 85%, 85%, 91%, respectively. The proposed CAT system thus establishes a noninvasive framework for monitoring cancer treatment response in tumors using clinical ultrasound imaging in conjunction with machine learning techniques. Such a framework can potentially facilitate the detection of refractory responses in patients to treatment early on during a course of therapy to enable possibly switching to more efficacious treatments.

Investigating longitudinal changes in the mechanical properties of MCF-7 cells exposed to paclitaxol using particle tracking microrheology

Evidence suggests that compression and shear wave elastography are sensitive to the mechanical property changes occuring in dying cells following chemotherapy, and can hence be used to monitor cancer treatment response. A qualitative and quantitative understanding of the mechanical changes at the cellular level would allow to better infer how these changes affect macroscopic tissue mechanical properties and therefore allow the optimization of elastographic techniques (such as shear wave elastography) for the monitoring of cancer therapy. We used intracellular particle tracking microrheology (PTM) to investigate the mechanical property changes of cells exposed to paclitaxol, a mitotic inhibitor used in cancer chemotherapy. The average elastic and viscous moduli of the cytoplasm of treated MCF-7 breast cancer cells were calculated for frequency ranges between 0.2 and 100 rad s–1 (corresponding to 0.03 and 15.92 Hz, respectively). A significant increase in the complex shear modulus of the cell cytoplasm was detected at 12 h post treatment. At 24 h after drug exposure, the elastic and viscous moduli increased by a total of 191.3 Pa (>8000×) and 9 Pa (∼9×), respectively for low frequency shear modulus measurements (at 1 rad s–1). At higher frequencies (10 rad s–1), the elastic and viscous moduli increased by 188.5 Pa (∼60×) and 1.7 Pa (∼1.1×), respectively. Our work demonstrates that PTM can be used to measure changes in the mechanical properties of treated cells and that cell elasticity significantly increases by 24 h after chemotherapy exposure.

Predicting and monitoring cancer treatment response with diffusion‐weighted MRI

Predicting and monitoring cancer treatment response with diffusion‐weighted MRI

Predicting and monitoring cancer treatment response with diffusion‐weighted MRI

An imaging biomarker that would provide for an early quantitative metric of clinical treatment response in cancer patients would provide for a paradigm shift in cancer care. Currently, nonimage based clinical outcome metrics include morphology, clinical, and laboratory parameters, however, these are obtained relatively late following treatment. Diffusion-weighted MRI (DW-MRI) holds promise for use as a cancer treatment response biomarker as it is sensitive to macromolecular and microstructural changes which can occur at the cellular level earlier than anatomical changes during therapy. Studies have shown that successful treatment of many tumor types can be detected using DW-MRI as an early increase in the apparent diffusion coefficient (ADC) values. Additionally, low pretreatment ADC values of various tumors are often predictive of better outcome. These capabilities, once validated, could provide for an important opportunity to individualize therapy thereby minimizing unnecessary systemic toxicity associated with ineffective therapies with the additional advantage of improving overall patient health care and associated costs. In this report, we provide a brief technical overview of DW-MRI acquisition protocols, quantitative image analysis approaches and review studies which have implemented DW-MRI for the purpose of early prediction of cancer treatment response.