External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts.

BACKGROUND: It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma. METHODS: RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs. RESULTS: In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner. CONCLUSIONS: We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

Phase II Trial Assessing the Repeatability and Tumor Uptake of [68Ga]Ga-HER2 Single-Domain Antibody PET/CT in Patients with Breast Carcinoma.

Human epidermal growth factor receptor 2 (HER2) status is used for decision-making in breast carcinoma treatment. The status is obtained through immunohistochemistry or in situ hybridization. These two methods have the disadvantage of necessitating tissue sampling, which is prone to error due to tumor heterogeneity or interobserver variability. Whole-body imaging might be a solution to map HER2 expression throughout the body. Methods: Twenty patients with locally advanced or metastatic breast carcinoma (5 HER2-positive and 15 HER2-negative patients) were included in this phase II trial to assess the repeatability of uptake quantification and the extended safety of the [68Ga]Ga-NOTA-anti-HER2 single-domain antibody (sdAb). The tracer was injected, followed by a PET/CT scan at 90 min. Within 8 d, the procedure was repeated. Blood samples were taken for antidrug antibody (ADA) assessment and liquid biopsies. On available tissues, immunohistochemistry, in situ hybridization, and mass spectrometry were performed to determine the correlation of HER2 status with uptake values measured on PET. If relevant preexisting [18F]FDG PET/CT images were available (performed as standard of care), a comparison was made. Results: With a repeatability coefficient of 21.8%, this imaging technique was repeatable. No clear correlation between PET/CT uptake values and pathology could be established, as even patients with low levels of HER2 expression showed moderate to high uptake. Comparison with [18F]FDG PET/CT in 16 patients demonstrated that in 7 patients, [68Ga]Ga-NOTA-anti-HER2 shows interlesional heterogeneity within the same patient, and [18F]FDG uptake did not show the same heterogeneous uptake in all patients. In some patients, the extent of disease was clearer with the [68Ga]Ga-NOTA-anti-HER2-sdAb. Sixteen adverse events were reported but all without a clear relationship to the tracer. Three patients with preexisting ADAs did not show adverse reactions. No new ADAs developed. Conclusion: [68Ga]Ga-NOTA-anti-HER2-sdAb PET/CT imaging shows similar repeatability to [18F]FDG. It is safe for clinical use. There is tracer uptake in cancer lesions, even in patients previously determined to be HER2-low or -negative. The tracer shows potential in the assessment of interlesional heterogeneity of HER2 expression. In a subset of patients, [68Ga]Ga-NOTA-anti-HER2-sdAb uptake was seen in lesions with no or low [18F]FDG uptake. These findings support further clinical development of [68Ga]Ga-NOTA-anti-HER2-sdAb as a PET/CT tracer in breast cancer patients.

Singlet oxygen-based photoelectrochemical detection of single-point mutations in the KRAS oncogene.

Single nucleotide point mutations in the KRAS oncogene occur frequently in human cancers, rendering them intriguing targets for diagnosis, early detection and personalized treatment. Current detection methods are based on polymerase chain reaction, sometimes combined with next-generation sequencing, which can be expensive, complex and have limited availability. Here, we propose a novel singlet oxygen (1O2)-based photoelectrochemical detection methodology for single-point mutations, using KRAS mutations as a case study. This detection method combines the use of a sandwich assay, magnetic beads and robust chemical photosensitizers, that need only air and light to produce 1O2, to ensure high specificity and sensitivity. We demonstrate that hybridization of the sandwich hybrid at high temperatures enables discrimination between mutated and wild-type sequences with a detection rate of up to 93.9%. Additionally, the presence of background DNA sequences derived from human cell-line DNA, not containing the mutation of interest, did not result in a signal, highlighting the specificity of the methodology. A limit of detection as low as 112 pM (1.25 ng/mL) was achieved without employing any amplification techniques. The developed 1O2-based photoelectrochemical methodology exhibits unique features, including rapidity, ease of use, and affordability, highlighting its immense potential in the field of nucleic acid-based diagnostics.

Belgian Recommendations for Analytical Verification and Validation of Immunohistochemical Tests in Laboratories of Anatomic Pathology.

Analytical verification and validation of immunohistochemical (IHC) tests and their equipment are common practices for today's anatomic pathology laboratories. Few references or guidelines are available on how this should be performed. The study of Sciensano (the Belgian national competent authority regarding licensing of medical laboratories) performed in 2016, demonstrated a significant interlaboratory variation in validation procedures of IHC tests among Belgian laboratories. These results suggest the unavailability of practical information on the approach to the verification and validation of these tests. The existing Belgian Practice Guideline for the implementation of a quality management system in anatomic pathology laboratories has been reviewed to meet this demand and, in addition, to prepare the laboratories for the EU-IVD revised regulations (IVDR). This paper describes Belgian recommendations for the verification and validation of IHC tests before implementation, for ongoing validation, and for revalidation. For each type of test (according to the IVDR classification and the origin) and its intended use (purpose), it addresses how to perform analytical verification/validation by recommending: (1) the number of cases in the validation set, (2) the performance characteristics to be evaluated, (3) the objective acceptance criteria, (4) the evaluation method for the obtained results, and (5) how and when to revalidate. A literature study and a risk analysis taking into account the majority of variables regarding verification/validation of methods have been performed, resulting in an expert consensus recommendation that is a compromise among achievability, affordability, and patient safety. This new consensus recommendation has been incorporated in the aforementioned ISO 15189:2012-based Practice Guideline.

PD-1, PD-L1, IDO, CD70 and microsatellite instability as potential targets to prevent immune evasion in sarcomas.

Background: Soft tissue and bone sarcomas are rare entities, hence, standardized therapeutic strategies are difficult to assess. Materials & methods: Immunohistochemistry was performed on 68 sarcoma samples to assess the expression of PD-1, PD-L1, IDO and CD70 in different tumor compartments and molecular analysis was performed to assess microsatellite instability status. Results: PD-1/PD-L1, IDO and CD70 pathways are at play in the immune evasion of sarcomas in general. Soft tissue sarcomas more often show an inflamed phenotype compared with bone sarcomas. Specific histologic sarcoma types show high expression levels of different markers. Finally, this is the first presentation of a microsatellite instability-high Kaposi sarcoma. Discussion/conclusion: Immune evasion occurs in sarcomas. Specific histologic types might benefit from immunotherapy, for which further investigation is needed.

Sarcomas of the soft tissue and bone are rare cancers. When these cancers spread to other parts of the body, it is hard to find good treatments. Recently, doctors have been using a new type of treatment called immunotherapy to fight several types of cancer. Immunotherapy works by getting one's body's own defense cells to attack the cancer cells. Unfortunately, immunotherapy does not work well for sarcomas and we do not know why. This study was designed to determine if there are certain mechanisms in these tumors that help the cancer cells to hide from defense cells. Determining how to change these mechanisms could make immunotherapy a better treatment for sarcomas in the future.

The immune microenvironment characterisation and dynamics in hormone receptor-positive breast cancer before and after neoadjuvant endocrine therapy.

BACKGROUND: Oestrogen receptor positive (ER+)/HER-2 negative breast cancer (BC) is considered to be an immunologically cold tumour compared to triple negative breast cancer. Therefore, the tumour microenvironment (TME) of ER+/HER-2 negative BC is understudied. The aim of this project is to investigate the TME and the immune response during neoadjuvant endocrine therapy (NET) and to correlate this with the treatment response in a real life setting. METHODS: Expression of immune checkpoint receptors and immune cells was examined immunohistochemically, pre- and post-NET in a cohort of 56 ER+/HER-2 negative BC patients. They were treated with tamoxifen (n = 16), an aromatase inhibitor (n = 40) or a combination of an aromatase inhibitor with a PI3K inhibitor (n = 11) for a median duration of 6 months (range 1-32 months). Immunohistochemical staining with monoclonal antibodies for PDL-1, PD-1, TIM-3, LAG-3, CTLA-4, CD4, CD68 and FOXP3 were performed. All staining procedures were done according to validated protocols, and scoring was done by a pathologist specialized in breast cancer. Positivity was defined as staining >1% on TILs. Response to NET was evaluated according to tumour size change on imaging and Ki-67 change. RESULTS: The median age was 61.02 (37-90) years. Diameter of tumour size decreased with a mean of 8.1 mm (-16 mm to 45 mm) (p < 0.001) during NET and the value of Ki-67 value decreased with a median of 9 after NET (p < 0.001). An increase in PD-L1 expression after NET showed a trend towards significant (p = 0.088) and CD-4+ T cells significantly increased after NET (p = 0.03). A good response to NET defined as a decrease in tumour size and/or decrease of Ki-67 was found to be associated with a longer duration of NET, a change of CD4+ T-cells and a higher number of CD68+ tumour-associated macrophages before the start of NET. CONCLUSION: The immune microenvironment plays an important role in ER+/HER-2 negative BC. NET influences the composition and functional state of the infiltrating immune cells. Furthermore, changes in the immune microenvironment are also associated with treatment response.

UAS™-A Urine Preservative for Oncology Applications.

Liquid biopsy is a revolutionary tool that is gaining momentum in the field of cancer research. As a body fluid, urine can be used in non-invasive diagnostics for various types of cancer. We investigated the performance of UAS™ as a preservative for urinary analytes. Firstly, the need for urine preservation was investigated using urine samples from healthy volunteers. Secondly, the performance of UAS™ was assessed for cell-free DNA (cfDNA) and host cell integrity during storage at room temperature (RT) and after freeze-thaw cycling. Finally, UAS™ was used in a clinical setting on samples from breast and prostate cancer patients. In the absence of a preservative, urinary cfDNA was degraded, and bacterial overgrowth occurred at RT. In urine samples stored in UAS™, no microbial growth was seen, and cfDNA and cellular integrity were maintained for up to 14 days at RT. After freeze-thaw cycling, the preservation of host cell integrity and cfDNA showed significant improvements when using UAS™ compared to unpreserved urine samples. Additionally, UAS™ was found to be compatible with several commercially available isolation methods.

Urine biomarkers in cancer detection: A systematic review of preanalytical parameters and applied methods.

The aim of this review was to explore the status of urine sampling as a liquid biopsy for noninvasive cancer research by reviewing used preanalytical parameters and protocols. We searched two main health sciences databases, PubMed and Web of Science. From all eligible publications (2010-2022), information was extracted regarding: (a) study population characteristics, (b) cancer type, (c) urine preanalytics, (d) analyte class, (e) isolation method, (f) detection method, (g) comparator used, (h) biomarker type, (i) conclusion and (j) sensitivity and specificity. The search query identified 7835 records, of which 924 unique publications remained after screening the title, abstract and full text. Our analysis demonstrated that many publications did not report information about the preanalytical parameters of their urine samples, even though several other studies have shown the importance of standardization of sample handling. Interestingly, it was noted that urine is used for many cancer types and not just cancers originating from the urogenital tract. Many different types of relevant analytes have been shown to be found in urine. Additionally, future considerations and recommendations are discussed: (a) the heterogeneous nature of urine, (b) the need for standardized practice protocols and (c) the road toward the clinic. Urine is an emerging liquid biopsy with broad applicability in different analytes and several cancer types. However, standard practice protocols for sample handling and processing would help to elaborate the clinical utility of urine in cancer research, detection and disease monitoring.

NPY Methylated ctDNA is a Promising Biomarker for Treatment Response Monitoring in Metastatic Colorectal Cancer.

PURPOSE: Analysis of methylation markers in liquid biopsies is a promising technique for the follow-up of patients with metastatic colorectal cancer (mCRC), because they can be used in all patients, regardless of their mutational status. Therefore, we studied the value of NPY methylation analysis in circulating tumor DNA (ctDNA) for accurate response monitoring in patients with mCRC in the PANIB trial. EXPERIMENTAL DESIGN: The PANIB trial was a randomized phase II trial designed to compare FOLFOX plus panitumumab and FOLFOX plus bevacizumab in patients with RAS wild-type unresectable mCRC. The results of sequential liquid biopsies were correlated with results of imaging. RESULTS: Forty patients were included from six Belgian hospitals. Analysis of the liquid biopsies revealed that higher baseline levels of methylated ctDNA was associated with a significantly shorter overall survival [HR, 1.015; 95% confidence interval (CI), 1.005-1.025; P = 0.002]. Furthermore, 37 patients provided at least two liquid biopsies. Thirty-one of them showed a decrease in the methylation ratio after the start of therapy, which corresponded with stable disease or response on imaging at the first evaluation. When comparing the panitumumab and bevacizumab arm, significantly higher objective response and early tumor shrinkage rates were observed in the panitumumab arm (P = 0.048 and 0.015, respectively). However, due to a small study population, the trial was underpowered to detect a significant difference in survival. CONCLUSIONS: The results of this study confirm that baseline methylated ctDNA is a prognostic marker and indicate that NPY methylation is a promising marker for response monitoring in patients with mCRC.

Molecular pathology testing for non-small cell lung cancer: an observational study of elements currently present in request forms and result reports and the opinion of different stakeholders.

BACKGROUND: For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. METHODS: This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. RESULTS: Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. CONCLUSIONS: Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

Are anaplastic lymphoma kinase (ALK) and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation driver biomarkers of pulmonary neuroendocrine tumors (NETs) and carcinomas (NECs)?

BACKGROUND: Novel targets in neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) are needed to improve outcome. The presence of O6-Methylguanine-DNA methyltransferase (MGMT) promoter methylation in NETs and NECs may act as a predictive marker for response on treatment with temozolomide. As anaplastic lymphoma kinase (ALK) plays an important role in the nervous system we hypothesized that ALK rearrangement can act as a biomarker in patients with NETs and NECs. MATERIALS AND METHODS: We performed a retrospective analysis to establish the frequency of MGMT promoter methylation and ALK expression in tissue samples of patients with NETs and NECs. RESULTS: 21% (14/67) of patients tested positive for MGMT promoter methylation. MGMT promoter methylation was present in 33% (3/9) patients with typical carcinoid, in 22% (2/9) patients with atypical carcinoid, in 22% (8/37) patients with small cell lung cancer and in 8% (1/12) patient with large cell neuroendocrine carcinoma. ALK- expression was present in 14% (10 of 70 patients). In all of these patients, no ALK-rearrangement nor ALK-mutation was revealed. CONCLUSIONS: Routine testing of NET and NEC samples for an ALK rearrangement is not recommended as ALK-expression is not associated with an ALK-rearrangement. Routine testing of NET and NEC samples for MGMT will detect a promoter hypermethylation in a sizable minority of patients who are eligible for a targeted treatment with temozolomide.

NTRK Gene Fusion Detection in a Pan-Cancer Setting Using the Idylla GeneFusion Assay.

Recently, approval of tyrosine receptor kinase (TRK) inhibitors by Food and Drug Administration and European Medicines Agency in NTRK fusion-positive cancer types has led to a variety of proposed testing algorithms. In this study, performance of the fully automated Idylla GeneFusion Assay was assessed in a set of clinically relevant cancer types, including glioblastoma, non-small-cell lung cancer, microsatellite instability-positive colorectal cancer, and thyroid carcinoma. Analysis with the Idylla GeneFusion Assay revealed significant differences in baseline RNA expression profile between the different cancer types, which corresponded to both literature and pan-TRK immunohistochemical staining. Compared with the RNA-based Oncomine Focus Assay, the Idylla GeneFusion Assay demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 92.7%, 81.8%, and 93.8%, respectively; and the pan-TRK immunohistochemistry demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 82.1%, 45.5%, and 85.7%, respectively. These findings highlighted the importance of tailoring NTRK testing algorithms per cancer type. In a small subset, data from the RNA-based Archer FusionPlex Assay were also available. NTRK fusion detection efficiency was compared between the four NTRK testing modalities, with a high concordance between the PCR-based methods. Last, RNA degradation was observed when using the Idylla GeneFusion Assay on snap frozen tissue samples as these are nonfixated. This might be countered by increasing the amount of sample input. To conclude, the Idylla GeneFusion Assay has shown a clear potential in identifying NTRK fusions.

Excellent Response to MEK Inhibition in an AGK-BRAF Gene Fusion Driven Carcinoma: Case Report and Literature Review.

BACKGROUND: Soft tissue myoepithelial carcinomas (STMC) are a rare, malignant subgroup of myoepithelial tumors that arise typically in glandular or ductal tissues, but also in the bone and soft and cutaneous tissues. Due to its rarity, there is no consensus regarding the treatment of STMC, including chemotherapy or other systemic agents for metastatic STMC. CASE REPORT: A chemotherapy- and regorafenib-refractory STMC, harboring an AGK-BRAF fusion, was successfully treated using MEK-inhibition with cobimetinib in monotherapy. MEK-inhibition with cobimetinib effectively silenced paradoxical MAP kinase/ERK-signaling pathway activation after regorafenib monotherapy, and resulted in a significant and durable clinical response. CONCLUSION: This effect of MEK-inhibition in STMC harboring an AGK-BRAF fusion has not been previously reported and contributes to the existing, yet limited, knowledge on the treatment of BRAF fusion-driven tumors. Also, our case highlights the importance of next generation sequencing in driving further rational therapeutic choices to provide disease control and palliation.

Cusatuzumab for treatment of CD70-positive relapsed or refractory cutaneous T-cell lymphoma.

BACKGROUND: The clinical benefit of cusatuzumab, a CD70-directed monoclonal antibody with enhanced effector functions, was investigated in patients with relapsed/refractory (R/R) cutaneous T-cell lymphoma (CTCL). METHODS: In this cohort expansion of the ARGX-110-1201 study, 27 patients with R/R CTCL received cusatuzumab at 1 (n = 11) or 5 mg/kg (n = 16) once every 3 weeks to investigate its safety, dose, and exploratory efficacy. The pharmacokinetics, immunogenicity, CD70 expression, and CD70/CD27 biology were also assessed. RESULTS: The most common adverse events included infusion-related reactions, pyrexia, and asthenia. Eighteen serious adverse events (grade 1-3) were reported in 11 patients; 1 of these (vasculitis) was considered drug-related. For 8 of the 11 patients receiving 1 mg/kg, anti-drug antibodies (ADAs) affected the minimal concentration, and this resulted in undetectable cusatuzumab concentrations at the end of treatment and, in some cases, a loss of response. This effect was greatly reduced in the patients receiving 5 mg/kg. The overall response rate was 23%; this included 1 complete response and 5 partial responses (PRs) in 26 of the 27 evaluable patients. In addition, 9 patients achieved stable disease. The mean duration on cusatuzumab was 5.2 months, and the median duration was 2.5 months. Patients with Sézary syndrome (SS) achieved a 60% PR rate with a dosage of 5 mg/kg and a 33% PR rate with a dosage of 1 mg/kg; this resulted in an overall response rate of 50% for patients with SS at both doses. CONCLUSIONS: Cusatuzumab was well tolerated, and antitumor activity was observed at both 1 and 5 mg/kg in highly pretreated patients with R/R CTCL. The observed dose-dependent effect on exposure supports the use of 5 mg/kg for future development.

Evaluation of Cytologic Sample Preparations for Compatibility With Nucleic Acid Analysis.

OBJECTIVES: In this study, the influence of several key elements of the cytologic sample workflow on DNA and RNA content was evaluated. METHODS: The A549 cell line, patient-derived organoids, and pleural effusions were used to investigate the effect of (1) several collection media and delayed time to processing; (2) cytology specimens; (3) cytologic staining; and (4) formalin-fixed, paraffin-embedded (FFPE) cell block processing on nucleic acid quality and quantity as determined by fragment analyzer, Qubit analysis (Thermo Fisher Scientific), and quantitative polymerase chain reaction-based analysis on the Idylla platform (Biocartis). RESULTS: Alcohol-based collection media (CytoRich Red [Thermo Fisher Scientific] and EtOH95%) displayed high DNA and RNA preservation capacity, while phosphate-buffered saline and, to a lesser extent, formalin were associated with high RNA quality. Cytospin and smear cytology specimens were subject to DNA and RNA loss. Cytologic staining had no further impact on sample quality, hence destaining is not necessary. Both H&E-stained and unstained FFPE sections are compatible with nucleic acid analysis, despite a strong decrease in DNA and RNA quality. CONCLUSIONS: Although several key elements of the cytologic sample workflow have an influence on DNA and RNA quality and quantity, the selection of these elements is also dependent on the downstream (ancillary) testing methods.

Immuno-PET Molecular Imaging of RANKL in Cancer.

PURPOSE: The involvement of RANK/RANKL signaling in the tumor microenvironment (TME) in driving response or resistance to immunotherapy has only very recently been recognized. Current quantification methods of RANKL expression suffer from issues such as sensitivity, variability, and uncertainty on the spatial heterogeneity within the TME, resulting in conflicting reports on its reliability and limited use in clinical practice. Non-invasive molecular imaging using immuno-PET is a promising approach combining superior targeting specificity of monoclonal antibodies (mAb) and spatial, temporal and functional information of PET. Here, we evaluated radiolabeled anti-RANKL mAbs as a non-invasive biomarker of RANKL expression in the TME. EXPERIMENTAL DESIGN: Anti-human RANKL mAbs (AMG161 and AMG162) were radiolabeled with 89Zr using the bifunctional chelator DFO in high yield, purity and with intact binding affinity. After assessing the biodistribution in healthy CD-1 nude mice, [89Zr]Zr-DFO-AMG162 was selected for further evaluation in ME-180 (RANKL-transduced), UM-SCC-22B (RANKL-positive) and HCT-116 (RANKL-negative) human cancer xenografts to assess the feasibility of in vivo immuno-PET imaging of RANKL. RESULTS: [89Zr]Zr-DFO-AMG162 was selected as the most promising tracer for further validation based on biodistribution experiments. We demonstrated specific accumulation of [89Zr]Zr-DFO-AMG162 in RANKL transduced ME-180 xenografts. In UM-SCC-22B xenograft models expressing physiological RANKL levels, [89Zr]Zr-DFO-AMG162 imaging detected significantly higher signal compared to control [89Zr]Zr-DFO-IgG2 and to RANKL negative HCT-116 xenografts. There was good visual agreement with tumor autoradiography and immunohistochemistry on adjacent slides, confirming these findings. CONCLUSIONS: [89Zr]Zr-DFO-AMG162 can detect heterogeneous RANKL expression in the TME of human cancer xenografts, supporting further translation of RANKL immuno-PET to evaluate tumor RANKL distribution in patients.

Prognostic and Predictive Biomarkers in Non-Small Cell Lung Cancer Patients on Immunotherapy-The Role of Liquid Biopsy in Unraveling the Puzzle.

In the last decade, immunotherapy has been one of the most important advances in the non-small cell lung cancer (NSCLC) treatment landscape. Nevertheless, only a subset of NSCLC patients benefits from it. Currently, the only Food and Drug Administration (FDA) approved diagnostic test for first-line immunotherapy in metastatic NSCLC patients uses tissue biopsies to determine the programmed death ligand 1 (PD-L1) status. However, obtaining tumor tissue is not always feasible and puts the patient at risk. Liquid biopsy, which refers to the tumor-derived material present in body fluids, offers an alternative approach. This less invasive technique gives real-time information on the tumor characteristics. This review addresses different promising liquid biopsy based biomarkers in NSCLC patients that enable the selection of patients who benefit from immunotherapy and the monitoring of patients during this therapy. The challenges and the opportunities of blood-based biomarkers such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), exosomes, epigenetic signatures, microRNAs (miRNAs) and the T cell repertoire will be addressed. This review also focuses on the less-studied feces-based and breath-based biomarkers.

Immune Checkpoint Inhibitory Therapy in Sarcomas: Is There Light at the End of the Tunnel?

Soft tissue and bone sarcomas are a very heterogeneous group of tumors with many subtypes for which diagnosis and treatment remains a very challenging task. On top of that, the treatment choices are limited, and the prognosis of aggressive sarcomas remains poor. Immune checkpoint inhibitors (ICIs) have drawn a lot of attention last years because of their promising response rates and their durable effects. ICIs are currently widely used in the daily routine practice for the treatment of a different malignancies, such as melanoma, Hodgkin lymphoma, and non-small cell lung carcinoma. Still, ICIs are not included in the standard treatment protocols of the different sarcoma types. However, a plethora of clinical trials investigates the clinical benefit of ICIs in sarcomas. There is clear need to develop predictive biomarkers to determine which sarcoma patients are most likely to benefit from immune checkpoint blockade. This review will focus on (i) the clinical trial results on the use of ICIs in different sarcoma types; and on (ii) possible biomarkers predictive for the effectiveness of these drugs in sarcomas.

Immunohistochemistry as a screening tool for NTRK gene fusions: results of a first Belgian ring trial.

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.