Advancing Evidence Generation for Circulating Tumor DNA: Lessons Learned from A Multi-Assay Study of Baseline Circulating Tumor DNA Levels across Cancer Types and Stages.

Circulating tumor DNA (ctDNA) holds promise as a biomarker for predicting clinical responses to therapy in solid tumors, and multiple ctDNA assays are in development. However, the heterogeneity in ctDNA levels prior to treatment (baseline) across different cancer types and stages and across ctDNA assays has not been widely studied. Friends of Cancer Research formed a collaboration across multiple commercial ctDNA assay developers to assess baseline ctDNA levels across five cancer types in early- and late-stage disease. This retrospective study included eight commercial ctDNA assay developers providing summary-level de-identified data for patients with non-small cell lung cancer (NSCLC), bladder, breast, prostate, and head and neck squamous cell carcinoma following a common analysis protocol. Baseline ctDNA levels across late-stage cancer types were similarly detected, highlighting the potential use of ctDNA as a biomarker in these cancer types. Variability was observed in ctDNA levels across assays in early-stage NSCLC, indicative of the contribution of assay analytical performance and methodology on variability. We identified key data elements, including assay characteristics and clinicopathological metadata, that need to be standardized for future meta-analyses across multiple assays. This work facilitates evidence generation opportunities to support the use of ctDNA as a biomarker for clinical response.

Accelerating the development of genetically engineered cellular therapies: a framework for extrapolating data across related products.

BACKGROUND: Significant advancements have been made in the field of cellular therapy as anti-cancer treatments, with the approval of chimeric antigen receptor (CAR)-T cell therapies and the development of other genetically engineered cellular therapies. CAR-T cell therapies have demonstrated remarkable clinical outcomes in various hematological malignancies, establishing their potential to change the current cancer treatment paradigm. Due to the increasing importance of genetically engineered cellular therapies in the oncology treatment landscape, implementing strategies to expedite development and evidence generation for the next generation of cellular therapy products can have a positive impact on patients. METHODS: We outline a risk-based methodology and assessment aid for the data extrapolation approach across related genetically engineered cellular therapy products. This systematic data extrapolation approach has applicability beyond CAR-T cells and can influence clinical development strategies for a variety of immune therapies such as T cell receptor (TCR) or genetically engineered and other cell-based therapies (e.g., tumor infiltrating lymphocytes, natural killer cells and macrophages). RESULTS: By analyzing commonalities in manufacturing processes, clinical trial designs, and regulatory considerations, key learnings were identified. These insights support optimization of the development and regulatory approval of novel cellular therapies. CONCLUSIONS: The field of cellular therapy holds immense promise in safely and effectively treating cancer. The ability to extrapolate data across related products presents opportunities to streamline the development process and accelerate the delivery of novel therapies to patients.

A Study Design to Harmonize Patient-Reported Outcomes Across Data Sets.

PURPOSE: Using patient-reported outcomes (PROs) provides important insights from the patient's perspective and can be valuable to monitor and manage treatment-related adverse events during cancer treatment. Additionally, the digital administration of PROs (electronic PROs [ePROs]) provides real-time updates to clinical care teams on treatment-related symptoms in-between clinic visits. However, given the variability in the methodology and timing of the data collection, using and harmonizing these data across different systems remains challenging. Identifying data elements to capture and operating procedures for harmonization across ePRO tools will expedite efforts to generate relevant and robust data on use of ePRO data in clinical care. METHODS: Friends of Cancer Research assembled a consortium of project partners from key health care sectors to align on a framework for ePRO data capture across ePRO tools and assessment of the impact of ePRO data capture on patient outcomes. RESULTS: We identified challenges and opportunities to align ePRO data capture across ePRO tools and aligned on key data elements for assessing the impact of ePRO data capture on patient care and outcomes. Ultimately, we proposed a study protocol to leverage ePRO data for symptom and adverse event management to measure real-world effectiveness of ePRO tool implementation on patient care and outcomes. CONCLUSION: This work provides considerations for harmonizing ePRO data sets and a common framework to align across multiple ePRO tools to assess the value of ePROs for improving patient outcomes. Future efforts to interpret evidence and evaluate the impact of ePRO tools on patient outcomes will be aided by improved alignment across studies.

Retrospective analysis of cancer-specific gene expression panel for thyroid fine needle aspiration specimens.

BACKGROUND: While molecular testing is a promising strategy for preoperative assessment of cytologically indeterminate thyroid nodules, thyroid fine needle aspiration biopsy (FNA) presents unique challenges for molecular assays, including contaminating peripheral blood mononuclear cells (PBMC) and variable numbers of evaluable epithelial thyroid cells. Moreover, the newly recognized entity, noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), has added an additional challenge to the currently available molecular diagnostic platforms. New diagnostic tools are still needed to correctly distinguish benign and malignant thyroid nodules preoperatively. METHODS: Twenty-two transcript splice variants from 12 genes we previously identified as discriminating benign from malignant thyroid nodules were characterized in 80 frozen thyroid tumors from 8 histological subtypes. Isoforms detectable in PBMC were excluded, and the 5 most discriminating isoforms were further validated by real-time quantitative PCR (qPCR) on intraoperative FNA samples from 59 malignant tumors, 55 benign nodules, and 23 NIFTP samples. The qPCR threshold cycle values for each transcript were normalized to the thyrocyte-specific thyroid peroxidase isoform 1 (TPO1) and z-transformed. Receiver operating characteristic (ROC) analyses of the composite transcript scores were used to evaluate classification of thyroid FNAs by the 5-gene isoform expression panel. RESULTS: A molecular signature was developed by combining expression levels of specific isoforms of CDH3, FNDC4, HMGA2, KLK7, and PLAG1. FNAs containing at least 12-36 thyrocytes were sufficient for this assay. The 5-gene composite score achieved an area under the ROC curve (AUC) of 0.86 for distinguishing malignant from benign nodules, with a specificity of 91%, sensitivity of 75%, negative predictive value of 91%, and positive predictive value of 74%. CONCLUSION: Our newly developed 5-gene isoform expression panel distinguishes benign from malignant thyroid tumors and, may help distinguish benign from malignant thyroid nodules in the context of the new NIFTP subtype.

Characterization of TERT and BRAF copy number variation in papillary thyroid carcinoma: An analysis of the cancer genome atlas study.

Alterations in the genome, including mutations and copy number variation (CNV), can drive cancer progression. The Cancer Genome Atlas (TCGA) project studying papillary thyroid cancer (PTC) identified a number of recurrent arm-level copy number amplifications, some spanning genes that are also commonly mutated in thyroid cancer. Herein, we focus on the role of TERT and BRAF CNV in PTC, including its relation to mutation status, gene expression, and clinicopathological characteristics. Utilizing TCGA CNV data, we identified focal amplifications and deletions involving the TERT and BRAF loci. TERT amplifications are more frequent in later stage thyroid tumors; in contrast, BRAF amplifications are not associated with stage. Furthermore, TERT amplifications are more frequently found in tumors also harboring TERT mutations, the combination further increasing TERT expression. Conversely, BRAF amplifications are more frequently found in BRAF wildtype tumors, and are more common in the follicular subtype of PTC as well as classic PTCs associated with a high follicular component and a RAS-like expression profile (assessed by the BRAF/RAS score). This is the first study to examine the TCGA thyroid dataset for gene-level CNV of TERT and BRAF, and their relationship with mutation status, tumor type and tumor stage. Assessing the differences in patterns of TERT and BRAF amplifications in the context of the mutation status of these genes may provide insight into the differing roles CNV can play depending on tumor type, and may lead to a better understanding of cancer drivers in thyroid cancer.

Exploring the epigenetic regulation of telomerase reverse transcriptase (TERT) in human cancer cell lines.

Telomerase regulation, including TERT promoter methylation, has been of long-standing interest to cancer biologists. Rowland et al. have now vastly expanded their ongoing characterization of TERT promoter methylation in cancer cells, analyzing the methylation patterns of 833 cell lines from 23 human cancers. They document a highly conserved pattern of hypomethylation around the proximal promoter, as well as a more heterogeneous region of hypermethylation further upstream, both associated with active TERT expression in cancer cells. They further describe the interplay between activating TERT promoter mutations and allelic methylation and transcription patterns. This valuable dataset represents the most extensive characterization of TERT promoter methylation in cancer cells to date and will help guide the future study of transcriptional regulation of telomerase. Comment on: https://doi.org/10.1002/1878-0261.12786.

Telomerase Reverse Transcriptase (TERT) Regulation in Thyroid Cancer: A Review.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of the enzyme telomerase and is essential for telomerase activity. Upregulation of TERT expression and resulting telomerase activity occurs in the large majority of malignancies, including thyroid cancer. This upregulation results in continued cellular proliferation and avoidance of cellular senescence and cell death. In this review we will briefly introduce TERT and telomerase activity as it pertains to thyroid cancer and, highlight the effects of TERT on cancer cells. We will also explore in detail the different TERT regulatory strategies and how TERT is reactivated in thyroid cancer cells, specifically. These regulatory mechanisms include both activating single base pair TERT promoter mutations and epigenetic changes at the promoter, including changes in CpG methylation and histone modifications that affect chromatin structure. Further, regulation includes the allele-specific regulation of the TERT promoter in thyroid cancer cells harboring the TERT promoter mutation. These entail allele-specific transcriptional activator binding, DNA methylation, histone modifications, and mono-allelic expression of TERT. Lastly, TERT copy number alterations and alternative splicing are also implicated. Both amplifications of the TERT locus and increased full-length transcripts and decreased inactive and dominant negative isoforms result in active telomerase. Finally, the clinical significance of TERT in thyroid cancer is also reviewed.

Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines.

Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.