Interpretability of radiomics models is improved when using feature group selection strategies for predicting molecular and clinical targets in clear-cell renal cell carcinoma: insights from the TRACERx Renal study.

BACKGROUND: The aim of this work is to evaluate the performance of radiomics predictions for a range of molecular, genomic and clinical targets in patients with clear cell renal cell carcinoma (ccRCC) and demonstrate the impact of novel feature selection strategies and sub-segmentations on model interpretability. METHODS: Contrast-enhanced CT scans from the first 101 patients recruited to the TRACERx Renal Cancer study (NCT03226886) were used to derive radiomics classification models to predict 20 molecular, histopathology and clinical target variables. Manual 3D segmentation was used in conjunction with automatic sub-segmentation to generate radiomics features from the core, rim, high and low enhancing sub-regions, and the whole tumour. Comparisons were made between two classification model pipelines: a Conventional pipeline reflecting common radiomics practice, and a Proposed pipeline including two novel feature selection steps designed to improve model interpretability. For both pipelines nested cross-validation was used to estimate prediction performance and tune model hyper-parameters, and permutation testing was used to evaluate the statistical significance of the estimated performance measures. Further model robustness assessments were conducted by evaluating model variability across the cross-validation folds. RESULTS: Classification performance was significant (p < 0.05, H0:AUROC = 0.5) for 11 of 20 targets using either pipeline and for these targets the AUROCs were within ± 0.05 for the two pipelines, except for one target where the Proposed pipeline performance increased by > 0.1. Five of these targets (necrosis on histology, presence of renal vein invasion, overall histological stage, linear evolutionary subtype and loss of 9p21.3 somatic alteration marker) had AUROC > 0.8. Models derived using the Proposed pipeline contained fewer feature groups than the Conventional pipeline, leading to more straightforward model interpretations without loss of performance. Sub-segmentations lead to improved performance and/or improved interpretability when predicting the presence of sarcomatoid differentiation and tumour stage. CONCLUSIONS: Use of the Proposed pipeline, which includes the novel feature selection methods, leads to more interpretable models without compromising prediction performance. TRIAL REGISTRATION: NCT03226886 (TRACERx Renal).

Late-Stage Metastatic Melanoma Emerges through a Diversity of Evolutionary Pathways.

Understanding the evolutionary pathways to metastasis and resistance to immune-checkpoint inhibitors (ICI) in melanoma is critical for improving outcomes. Here, we present the most comprehensive intrapatient metastatic melanoma dataset assembled to date as part of the Posthumous Evaluation of Advanced Cancer Environment (PEACE) research autopsy program, including 222 exome sequencing, 493 panel-sequenced, 161 RNA sequencing, and 22 single-cell whole-genome sequencing samples from 14 ICI-treated patients. We observed frequent whole-genome doubling and widespread loss of heterozygosity, often involving antigen-presentation machinery. We found KIT extrachromosomal DNA may have contributed to the lack of response to KIT inhibitors of a KIT-driven melanoma. At the lesion-level, MYC amplifications were enriched in ICI nonresponders. Single-cell sequencing revealed polyclonal seeding of metastases originating from clones with different ploidy in one patient. Finally, we observed that brain metastases that diverged early in molecular evolution emerge late in disease. Overall, our study illustrates the diverse evolutionary landscape of advanced melanoma. SIGNIFICANCE: Despite treatment advances, melanoma remains a deadly disease at stage IV. Through research autopsy and dense sampling of metastases combined with extensive multiomic profiling, our study elucidates the many mechanisms that melanomas use to evade treatment and the immune system, whether through mutations, widespread copy-number alterations, or extrachromosomal DNA. See related commentary by Shain, p. 1294. This article is highlighted in the In This Issue feature, p. 1275.

Spatial patterns of tumour growth impact clonal diversification in a computational model and the TRACERx Renal study.

Genetic intra-tumour heterogeneity fuels clonal evolution, but our understanding of clinically relevant clonal dynamics remain limited. We investigated spatial and temporal features of clonal diversification in clear cell renal cell carcinoma through a combination of modelling and real tumour analysis. We observe that the mode of tumour growth, surface or volume, impacts the extent of subclonal diversification, enabling interpretation of clonal diversity in patient tumours. Specific patterns of proliferation and necrosis explain clonal expansion and emergence of parallel evolution and microdiversity in tumours. In silico time-course studies reveal the appearance of budding structures before detectable subclonal diversification. Intriguingly, we observe radiological evidence of budding structures in early-stage clear cell renal cell carcinoma, indicating that future clonal evolution may be predictable from imaging. Our findings offer a window into the temporal and spatial features of clinically relevant clonal evolution.

Determinants of anti-PD-1 response and resistance in clear cell renal cell carcinoma.

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action.

Specific Point-of-Care Testing of Coagulation in Patients Treated with Dabigatran.

BACKGROUND AND PURPOSE: Accurate and rapid assessment of coagulation status is necessary to guide thrombolysis or reversal of anticoagulation in stroke patients, but commercially available point-of-care (POC) assays are not suited for coagulation testing in patients treated with direct oral anticoagulants (DOACs). We aimed to evaluate the direct thrombin monitoring (DTM) test card by Helena Laboratories (Texas, United States) for anti-IIa-specific POC coagulation testing, hypothesizing that its POC-ecarin clotting time (POC-ECT) accurately reflects dabigatran plasma concentrations. METHODS: A prospective single-center diagnostic study (ClinicalTrials.gov-identifier: NCT02825394) was conducted enrolling patients receiving a first dose of dabigatran and patients already on dabigatran treatment. Blood samples were collected before drug intake and 0.5, 1, 2, 8, and 12 hours after intake. POC-ECT was performed using whole blood (WB), citrated blood (CB), and citrated plasma (CP). Dabigatran plasma concentrations were determined by mass spectrometry. RESULTS: In total, 240 blood samples from 40 patients contained 0 to 275 ng/mL of dabigatran. POC-ECT with WB/CB/CP ranged from 20 to 186/184/316 seconds. Pearson's correlation coefficient showed a strong correlation between dabigatran concentrations and POC-ECT with WB/CB/CP (R2 = 0.78/0.90/0.92). Dabigatran concentrations >30 and >50 ng/mL (thresholds for thrombolysis, surgery, and reversal therapy according to clinical guidelines) were detected by POC-ECT with WB/CB/CP (>36/35/45 and >43/45/59 seconds) with 95/97/97 and 96/98/97% sensitivity, and 81/87/94 and 74/60/91% specificity. CONCLUSION: This first study evaluating DOAC-specific POC coagulation testing revealed an excellent correlation of POC-ECT with actual dabigatran concentrations. Detecting clinically relevant dabigatran levels with high sensitivity/specificity, the DTM assay represents a suitable diagnostic tool in acute stroke, hemorrhage, and urgent surgery.

Limitations of Specific Coagulation Tests for Direct Oral Anticoagulants: A Critical Analysis.

Background During treatment with direct oral anticoagulants ( DOAC ), coagulation assessment is required before thrombolysis, surgery, and if anticoagulation reversal is evaluated. Limited data support the accuracy of DOAC -specific coagulation assays around the current safe-for-treatment threshold of 30 ng/ mL . Methods and Results In 481 samples obtained from 96 patients enrolled at a single center, DOAC concentrations were measured using Hemoclot direct thrombin inhibitor assay, Biophen direct thrombin inhibitor assay or ecarin clotting time for dabigatran, chromogenic anti-Xa assay ( AXA ) for factor Xa inhibitors (rivaroxaban, apixaban) and ultraperformance liquid chromatography-tandem mass spectrometry as reference. All dabigatran-specific assays had high sensitivity to concentrations >30 ng/ mL , but specificity was lower for Hemoclot direct thrombin inhibitor assay (78.2%) than for Biophen direct thrombin inhibitor assay (98.9%) and ecarin clotting time (94.6%). AXA provided high sensitivity and specificity for rivaroxaban, but low sensitivity for apixaban (73.8%; concentrations up to 82 ng/ mL were misclassified as <30 ng/ mL ). If no DOAC -specific calibration for AXA is available, results 2-fold above the upper limit of normal indicate relevant rivaroxaban concentrations. For apixaban, all elevated results should raise suspicion of relevant anticoagulation. Conclusions DOAC -specific tests differ considerably in diagnostic performance for concentrations close to the currently accepted safe-for-treatment threshold. Compared with Biophen direct thrombin inhibitor assay and ecarin clotting time, limited specificity of Hemoclot direct thrombin inhibitor assay poses a high risk of unnecessary anticoagulation reversal or treatment delays in patients on dabigatran. While AXA accurately detected rivaroxaban, the impact of low apixaban levels on the assay was weak. Hence, AXA results need to be interpreted with extreme caution when used to assess hemostatic function in patients on apixaban. Clinical Trial Registration URL : https://www.clinicaltrials.gov . Unique identifiers: NCT 02371044, NCT 02371070.

Diagnostic Accuracy of a Novel Chromogenic Direct Thrombin Inhibitor Assay: Clinical Experiences for Dabigatran Monitoring.

Background Direct oral anticoagulants (DOACs) are increasingly replacing vitamin K antagonists (VKA) for clinical indications requiring long-term oral anticoagulation. In contrast to VKA, treatment with DOAC including dabigatran­the only direct thrombin inhibitor amongst them­does not require therapeutic drug monitoring. However, in case of treatment complications (e.g., major haemorrhage) and conditions requiring urgent surgery or thrombolytic therapy, information about actual DOAC plasma levels is needed to guide treatment decisions. Due to short reagent stability, limited accuracy at low dabigatran levels and high heparin sensitivity, the applicability of the widely used Hemoclot thrombin inhibitor (HTI) coagulation assay is limited in the emergency setting. Methods Dabigatran concentrations of 288 citrated plasma samples taken from 48 dabigatran-treated patients with drug concentrations of up to 300 ng/mL were measured with the chromogenic anti-IIa Biophen direct thrombin inhibitor (BDTI) assay and results compared with HTI using ultra performance liquid chromatography­tandem mass spectrometry as the reference method for measuring dabigatran plasma concentrations. Results BDTI results showed a very strong correlation with dabigatran concentrations (r = 0.965, p < 0.0001) as well as a low intra- and inter-assay variation of <5%. Compared with HTI, BDTI provides an improved on-board reagent stability of 72 hours, rapid turnaround times comparable to routine coagulation assays, high accuracy at low drug levels and reduced heparin sensitivity. Conclusion The BDTI is an ideal coagulation assay for the around-the-clock determination of dabigatran plasma levels in clinical routine including emergency situations.

Point-of-care testing for emergency assessment of coagulation in patients treated with direct oral anticoagulants.

BACKGROUND: Point-of-care testing (POCT) of coagulation has been proven to be of great value in accelerating emergency treatment. Specific POCT for direct oral anticoagulants (DOAC) is not available, but the effects of DOAC on established POCT have been described. We aimed to determine the diagnostic accuracy of Hemochron® Signature coagulation POCT to qualitatively rule out relevant concentrations of apixaban, rivaroxaban, and dabigatran in real-life patients. METHODS: We enrolled 68 patients receiving apixaban, rivaroxaban, or dabigatran and obtained blood samples at six pre-specified time points. Coagulation testing was performed using prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (aPTT), and activated clotting time (ACT+ and ACT-low range) POCT cards. For comparison, laboratory-based assays of diluted thrombin time (Hemoclot) and anti-Xa activity were conducted. DOAC concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Four hundred and three samples were collected. POCT results of PT/INR and ACT+ correlated with both rivaroxaban and dabigatran concentrations. Insufficient correlation was found for apixaban. Rivaroxaban concentrations at <30 and <100 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.0 and ≤1.1 and ACT+ POCT ≤120 and ≤130 s. Dabigatran concentrations at <30 and <50 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.1 and ≤1.2 and ACT+ POCT ≤100 s. CONCLUSIONS: Hemochron® Signature POCT can be a fast and reliable alternative for guiding emergency treatment during rivaroxaban and dabigatran therapy. It allows the rapid identification of a relevant fraction of patients that can be treated immediately without the need to await the results of much slower laboratory-based coagulation tests. TRIAL REGISTRATION: Unique identifier, NCT02371070 . Retrospectively registered on 18 February 2015.

Involvement of transcription termination factor 2 in mitotic repression of transcription elongation.

All nuclear transcription is interrupted during mitosis. We examined the role of human TTF2, an RNA polymerase (Pol) I and II termination factor, in mitotic repression of transcription elongation. We find that TTF2 levels rise in the cytoplasm in S and G2 and at the onset of mitosis TTF2 translocates into the nucleus. Consistent with a role in termination of all transcription, TTF2 is the only ATP-dependent termination activity associated with Pol II transcription elongation complexes, is largely unaffected by template position, and is impervious to the phosphorylation state of the polymerase. Cells in which TTF2 levels are knocked down showed dramatic retention of Ser2 phosphorylated Pol II on mitotic chromosomes and an increase in chromosome segregation defects.