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Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors combined with endocrine therapy have transformed the treatment of estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2 negative (HER2-) metastatic breast cancer. However, some patients do not respond to this treatment, and patients inevitably develop resistance, such that novel biomarkers are needed to predict primary resistance, monitor treatment response for acquired resistance, and personalize treatment strategies. Circumventing the spatial and temporal limitations of tissue biopsy, newly developed liquid biopsy approaches have the potential to uncover biomarkers that can predict CDK4/6 inhibitor efficacy and resistance in breast cancer patients through a simple blood test. Studies on circulating tumor DNA (ctDNA)-based liquid biopsy biomarkers of CDK4/6 inhibitor resistance have focused primarily on genomic alterations and have failed thus far to identify clear and clinically validated predictive biomarkers, but emerging epigenetic ctDNA methodologies hold promise for further discovery. The present review outlines recent advances and future directions in ctDNA-based biomarkers of CDK4/6 inhibitor treatment response.

Supplementary File 4 shows transcriptional pathways associated with radioresponse within distinct tissue types.

Supplementary Figure from Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma

Supplementary File 2 shows transcriptional pathways associated with alpha or beta from the linear-quadratic formalism.

The Supplementary Data file contains 10 supplementary figures and 3 supplementary tables.

A schematic of tumor/host combinations showing Gal-1 WT and null C57Bl6/mice subcutaneous injection with Wt and shGal-1 LLC1 cells, time line of cell injection, tumor irradiation and blood collection time points. The relative plasma Gal-1 levels for each treatment group is also shown.

Supplementary materials and methods and figure legends from Galectin-1 Mediates Radiation-Related Lymphopenia and Attenuates NSCLC Radiation Response

Gal-1 inhibition increases tumor radiation response in vitro and in vivo.

Supplementary Data from HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Supplementary Figure from HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Supplementary Table 1. Patient characteristics of 20 NSCLC patient receiving SABR. Supplementary Table 2. Patient characteristics of 24 HNSCC patients receiving radiation with cetuximab or cisplatin.

Radiotherapy is integral to the care of a majority of patients with cancer. Despite differences in tumor responses to radiation (radioresponse), dose prescriptions are not currently tailored to individual patients. Recent large-scale cancer cell line databases hold the promise of unravelling the complex molecular arrangements underlying cellular response to radiation, which is critical for novel predictive biomarker discovery. Here, we present RadioGx, a computational platform for integrative analyses of radioresponse using radiogenomic databases. We fit the dose–response data within RadioGx to the linear-quadratic model. The imputed survival across a range of dose levels (AUC) was a robust radioresponse indicator that correlated with biological processes known to underpin the cellular response to radiation. Using AUC as a metric for further investigations, we found that radiation sensitivity was significantly associated with disruptive mutations in genes related to nonhomologous end joining. Next, by simulating the effects of different oxygen levels, we identified putative genes that may influence radioresponse specifically under hypoxic conditions. Furthermore, using transcriptomic data, we found evidence for tissue-specific determinants of radioresponse, suggesting that tumor type could influence the validity of putative predictive biomarkers of radioresponse. Finally, integrating radioresponse with drug response data, we found that drug classes impacting the cytoskeleton, DNA replication, and mitosis display similar therapeutic effects to ionizing radiation on cancer cell lines. In summary, RadioGx provides a unique computational toolbox for hypothesis generation to advance preclinical research for radiation oncology and precision medicine.Significance:The RadioGx computational platform enables integrative analyses of cellular response to radiation with drug responses and genome-wide molecular data.

Supplementary Data from Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma

Purpose:Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation.Experimental Design:We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I–IVA human papillomavirus–negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls.Results:CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples.Conclusions:Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

Tumor irradiation effect on T lymphocytes, common lymphoid progenitor cells (CLPs) and Gal-1 expression in bone marrow cells.

Supplementary File 1 shows transcriptional pathways associated with the radioresponse indicators SF2 or AUC.

Radiation-related lymphopenia is associated with worse distant, local and regional progression free survival in NSCLC, in similar between radiation and cetuximab or cisplatin treated HNSCC and its severity trends with relapse in HNSCC. A higher rise in plasma Gal-1 was also seen with relapse.

Purpose: Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities.Experimental Design: Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non–small cell lung cancer (NSCLC)] and head and neck cancer patients.Results: LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8+ T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients.Conclusions: Radiotherapy-related systemic lymphopenia appeared to be mediated by radiotherapy-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis. Clin Cancer Res; 20(21); 5558–69. ©2014 AACR.

Purpose:Human papillomavirus (HPV) DNA offers a convenient circulating tumor DNA (ctDNA) marker for HPV-associated malignancies, but current methods, such as digital PCR (dPCR), provide insufficient accuracy for clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings.Experimental Design:We conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability.Results:HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R2 = 0.95, P = 1.9 × 10–29) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non–cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes.Conclusions:HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. Our findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.

Increases in gal-1 secretion after tumor cell irradiation in vitro.

Lymphocytes from Gal-1 WT and null mice exhibiting similar sensitivity to ionizing radiation and recombinant Gal-1.

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PDF file - 52K, Clinical information for BCA patient-derived xenografts.

PDF file - 581K, Breast TICs are enriched in the CD24HiCD49fHi subpopulation (S1); Expression of HER receptors and ligands in breast cancer cell lines (S2); HER2 inhibition reduces the EpCAM- TIC-like subpopulation of SUM149 cells (S3); Breast TICs are enriched in the EPCAM+CD49f+ subpopulation of a triple-negative patient-derived xenograft breast tumor BCA70 (S4); Dual inhibition of HER2 and EGFR targets breast TICs in patient-derived xenograft BCA71 (S5).

IMPORTANCE: Patient-derived xenografts (PDXs) offer the opportunity to identify patients with oral cavity squamous cell carcinoma (OSCC) who are at risk for recurrence and optimize clinical decision-making. OBJECTIVE: To develop and validate a prediction score for locoregional failure (LRF) and distant metastases (DM) in OSCC that incorporates PDX engraftment in addition to known clinicopathological risk factors. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective cohort study, PDX models were generated from patients with OSCC treated with curative intent at Princess Margaret Cancer Centre (Toronto, Canada) between 2006 and 2018. The cohort included 288 patients (aged ≥18 years) with a new diagnosis of nonmetastatic (M0) OSCC whose tumor samples were available for engraftment under the skin of xenograft mice. Patients were scored as a nonengrafter if PDX formation did not occur within 6 months. Data analysis was performed between August 2006 and May 2018. INTERVENTIONS: All patients received up-front curative-intent surgery followed by either observation or postoperative radiation with or without concurrent chemotherapy based on institutional guidelines. MAIN OUTCOMES AND MEASURES: Main outcomes were LRF, DM, and overall survival (OS). Multivariable analysis (MVA) was used to identify predictors of LRF and DM. Factors retained in the final MVA were used to construct a prediction score and classify patients into risk groups. RESULTS: Overall, 288 patients (mean [SD] age at diagnosis, 63.3 [12.3] years; 112 [39%] women and 176 [61%] men) with OSCC were analyzed. The MVA identified pT3-4, pathologic extranodal extension, and engraftment as predictors of LRF and DM. Patients whose tumors engrafted (n = 198) were more likely to develop LRF (hazard ratio [HR], 1.98; 95% CI, 1.24-3.18) and DM (HR, 2.64; 95% CI, 1.21-5.75) compared with nonengrafters. A prediction score based on the aforementioned variables identified patients at high risk and low risk for LRF (43.5% vs 26.5%), DM (38.2% vs 8.4%), and inferior OS (34% vs 66%) at 5 years. Additionally, rapid engraftment was shown to be similarly prognostic, with rapid engrafters demonstrating higher rates of relapse and poor OS. CONCLUSIONS: In this cohort study, a prediction score using OSCC PDX engraftment, in conjunction with pT3-4 and pathologic extranodal extension, was associated with improved prognostic utility of existing clinical models and predicted patients at risk for LRF, DM, and poor survival.

Serial circulating tumor DNA (ctDNA) monitoring is emerging as a non-invasive strategy to predict and monitor immune checkpoint blockade (ICB) therapeutic efficacy across cancer types. Yet, limited data exist to show the relationship between ctDNA dynamics and tumor genome and immune microenvironment in patients receiving ICB. Here, we present an in-depth analysis of clinical, whole-exome, transcriptome, and ctDNA profiles of 73 patients with advanced solid tumors, across 30 cancer types, from a phase II basket clinical trial of pembrolizumab (NCT02644369) and report changes in genomic and immune landscapes (primary outcomes). Patients stratified by ctDNA and tumor burden dynamics correspond with survival and clinical benefit. High mutation burden, high expression of immune signatures, and mutations in BRCA2 are associated with pembrolizumab molecular sensitivity, while abundant copy-number alterations and B2M loss-of-heterozygosity corresponded with resistance. Upon treatment, induction of genes expressed by T cell, B cell, and myeloid cell populations are consistent with sensitivity and resistance. We identified the upregulated expression of PLA2G2D, an immune-regulating phospholipase, as a potential biomarker of adaptive resistance to ICB. Together, these findings provide insights into the diversity of immunogenomic mechanisms that underpin pembrolizumab outcomes., Although circulating tumour DNA (ctDNA) can predict immune checkpoint blockade (ICB) responses, its association with tumour biomarkers remains unknown. Here, the authors use ctDNA to inform exome and transcriptome profiling of >100 patients with 30 cancer types on a single clinical ICB trial and identify tumour microenvironment features associated with response.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA– a bona fide cancer biomarker in a subset of head and neck cancers– from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments

Introduction: Clinical benefit from Immune Checkpoint Blockade (ICB) is a function of local T-cell specificity for tumor-associated antigens. However, overcoming local T-cell dysfunction necessitates systemic immunity engagement. Therefore, studying the dynamics of both local and peripheral T-cell repertoires in response to ICB is required to identify features of T-cell repertoires associated with pathological response. Methods: We conducted TCRβ-sequencing on tumor-residing T-cells (n=59), Peripheral Blood Mononuclear Cells (PBMCs) (n=306) and cell-free DNA (cfDNA) (n=73) from pre- and multiple on-ICB timepoints collected from patients enrolled in the pan-cancer INvestigator-initiated Phase II Study of Pembrolizumab Immunological Response Evaluation (INSPIRE; NCT02644369) trial. To assess specificity-agnostic shifts in TCR repertoires, we first compared TCR diversity and clonal expansion in longitudinal tumor and PBMC samples. Then, to temporally track the specificity-associated features of local and systemic TCR repertoires, we leveraged a Graph Neural Network (GNN) model that took in unique TCRβ chains as nodes. The connectivity between the nodes was defined by multi-relational edges that represented VJ-gene usage and GLIPHII-identified (Grouping Lymphocyte Interactions by Paratope Hotspots) specificities derived from a compendium of TCR sequences with empirically confirmed specificities. Results: While absolute diversity and clonal expansion values in baseline tumor (n=33) were not associated with response to ICB, changes in these values were informative between pre- and on-ICB tumors. All patients (n=4) with low baseline tumor TCR diversity and lack of clonotypic re-structuring in tumor TCR repertoire on-ICB had either progressive or short-term stable disease. Furthermore, pairwise comparison of pre- and on-ICB tumors for each patient (n=17) revealed that all the patients, irrespective of their pathological response, experienced emergence of new TCR clonotypes (i.e., clonal replacement) in response to ICB, suggesting only a minority of these TCRs might consist of tumor-associated clonotypes. Patients with clinical benefit also had higher degree of GLIPHII-identified clustering at baseline tumor, highlighting the role of both specificity-agnostic and specificity-centric TCR analysis in determining the response to ICB. Analysis of TCR sequences in blood plasma found cfDNA contains a small number of TCR sequences (median 32, range 12-89) enriched for TCRs found in matched tumor tissues, suggesting that cfDNA TCR repertoire may provide an indirect measurement of tumor-residing T-cells. Conclusions: TCR diversity and functional clonal annotation are emerging biomarkers of ICB response and cfDNA TCR repertoire can potentially be exploited for clinical diagnostics and monitoring. Citation Format: Shirin Soleimani, Ben X. Wang, Stephanie Pedersen, Jenna Eagles, Jacob Brick, Marcus O. Butler, Scott V. Bratman, Lillian L. Siu, Pamela S. Ohashi, Trevor J. Pugh. Pan-cancer assessment of tumour and peripheral T-cell receptor repertoire dynamics in patients treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6667.