Single-stranded pre-methylated 5mC adapters uncover the methylation profile of plasma ultrashort Single-stranded cell-free DNA.

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

META RNA profiling: Multiplexed quantitation of targeted RNAs across large numbers of samples.

META RNA profiling is a simple and inexpensive method to measure the expression of multiple targeted RNAs across many samples. By assigning sample-specific tags up-front during reverse-transcription, cDNAs from multiple samples can be pooled prior to amplification and deep sequencing. Such early parallelization of samples simplifies the workflow, minimizes cross-sample experimental variability, and reduces reagent and sequencing costs. Herein we describe the theoretical framework of the method and provide a detailed protocol to facilitate its implementation.

High-throughput RNA profiling via up-front sample parallelization.

We describe a method called modular, early-tagged amplification (META) RNA profiling that can quantify a broad panel of microRNAs or mRNAs simultaneously across many samples and requires far less sequence depth than existing digital profiling technologies. The method assigns quantitative tags during reverse transcription to permit up-front sample pooling before competitive amplification and deep sequencing. This simple, scalable and inexpensive approach improves the practicality of large-scale gene expression studies.

Circulating Tumor DNA Dynamics Fail to Predict Efficacy of Poly(ADP-ribose) Polymerase/VEGFR Inhibition in Patients With Heavily Pretreated Advanced Solid Tumors.

PURPOSE: Cell-free circulating tumor DNA (ctDNA) has shown its potential as a quantitative biomarker for longitudinal monitoring of response to anticancer therapies. However, ctDNA dynamics have not been studied in patients with heavily pretreated, advanced solid tumors, for whom therapeutic responses can be weak. We investigated whether changes in ctDNA could predict clinical outcomes in such a cohort treated with combined poly(ADP-ribose) polymerase/vascular endothelial growth factor receptor inhibitor therapy. MATERIALS AND METHODS: Patients with metastatic pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), small-cell lung cancer (SCLC), or non-small-cell lung cancer (NSCLC) received up to 7 days of cediranib 30 mg orally once daily monotherapy lead-in followed by addition of olaparib 200 mg orally twice daily. Patients had progressed on a median of three previous lines of therapy. Plasma samples were collected before and after cediranib monotherapy lead-in and on combination therapy at 7 days, 28 days, and every 28 days thereafter. ctDNA was quantified from plasma samples using a multigene mutation-based assay. Radiographic assessment was performed every 8 weeks. RESULTS: ctDNA measurements were evaluable in 63 patients. The median baseline ctDNA variant allele fractions (VAFs) were 20%, 28%, 27%, and 34% for PDAC, TNBC, SCLC, and NSCLC, respectively. No association was observed between baseline VAF and radiographic response, progression-free survival, or overall survival (OS). Similarly, no association was found between ctDNA decline and radiographic response or survival. However, an increase in ctDNA at 56 days of combination therapy was associated with disease progression and inferior OS in a landmark analysis. CONCLUSION: ctDNA levels or dynamics did not correlate with radiographic response or survival outcomes in patients with advanced metastatic malignancies treated with olaparib and cediranib.

Linoleoyl-lysophosphatidylcholine suppresses immune-related adverse events due to immune checkpoint blockade.

Immune related adverse events (irAEs) after immune checkpoint blockade (ICB) therapy occur in a significant proportion of cancer patients. To date, the circulating mediators of ICB-irAEs remain poorly understood. Using non-targeted mass spectrometry, here we identify the circulating bio-active lipid linoleoyl-lysophosphatidylcholine (LPC 18:2) as a modulator of ICB-irAEs. In three independent human studies of ICB treatment for solid tumor, loss of circulating LPC 18:2 preceded the development of severe irAEs across multiple organ systems. In both healthy humans and severe ICB-irAE patients, low LPC 18:2 was found to correlate with high blood neutrophilia. Reduced LPC 18:2 biosynthesis was confirmed in preclinical ICB-irAE models, and LPC 18:2 supplementation in vivo suppressed neutrophilia and tissue inflammation without impacting ICB anti-tumor response. Results indicate that circulating LPC 18:2 suppresses human ICB-irAEs, and LPC 18:2 supplementation may improve ICB outcomes by preventing severe inflammation while maintaining anti-tumor immunity.

Platelet-derived TLT-1 promotes tumor progression by suppressing CD8+ T cells.

Current understanding of tumor immunosuppressive mechanisms forms the basis for modern day immunotherapies. Immunoregulatory role of platelets in cancer remains largely elusive. Platelets from non-small cell lung cancer (NSCLC) patients revealed a distinct activation phenotype. TREM-like transcript 1 (TLT-1), a platelet protein, was increased along with enhanced extracellular release from NSCLC platelets. The increased platelet TLT-1 was also evident in humanized mice with patient-derived tumors. In immunocompetent mice with syngeneic tumors, TLT-1 binding to T cells, in vivo, led to suppression of CD8 T cells, promoting tumor growth. We identified direct interaction between TLT-1 and CD3ε on T cells, implicating the NF-κB pathway in CD8 T cell suppression. Anti-TLT-1 antibody rescued patients' T cells from platelet-induced suppression ex vivo and reduced tumors in mice in vivo. Clinically, higher TLT-1 correlated with reduced survival of NSCLC patients. Our findings thus identify TLT-1 as a platelet-derived immunosuppressor that suppresses CD8 T cells and demonstrate its therapeutic and prognostic significance in cancer.

Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer.

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies.

The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring.

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.

Direct capture and sequencing reveal ultra-short single-stranded DNA in biofluids.

Cell-free DNA (cfDNA) has become the predominant analyte of liquid biopsy; however, recent studies suggest the presence of subnucleosomal-sized DNA fragments in circulation that are likely single-stranded. Here, we report a method called direct capture and sequencing (DCS) tailored to recover such fragments from biofluids by directly capturing them using short degenerate probes followed by single strand-based library preparation and next-generation sequencing. DCS revealed a new DNA population in biofluids, named ultrashort single-stranded DNA (ussDNA). Evaluation of the size distribution and abundance of ussDNA manifested generality of its presence in humans, animal species, and plants. In humans, red blood cells were found to contain abundant ussDNA; plasma-derived ussDNA exhibited modal size at 50 nt. This work reports the presence of an understudied DNA population in circulation, and yet more work is awaiting to study its generation mechanism, tissue of origin, disease implications, etc.

Limitations and opportunities of technologies for the analysis of cell-free DNA in cancer diagnostics.

Cell-free DNA (cfDNA) in the circulating blood plasma of patients with cancer contains tumour-derived DNA sequences that can serve as biomarkers for guiding therapy, for the monitoring of drug resistance, and for the early detection of cancers. However, the analysis of cfDNA for clinical diagnostic applications remains challenging because of the low concentrations of cfDNA, and because cfDNA is fragmented into short lengths and is susceptible to chemical damage. Barcodes of unique molecular identifiers have been implemented to overcome the intrinsic errors of next-generation sequencing, which is the prevailing method for highly multiplexed cfDNA analysis. However, a number of methodological and pre-analytical factors limit the clinical sensitivity of the cfDNA-based detection of cancers from liquid biopsies. In this Review, we describe the state-of-the-art technologies for cfDNA analysis, with emphasis on multiplexing strategies, and discuss outstanding biological and technical challenges that, if addressed, would substantially improve cancer diagnostics and patient care.

Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.

DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show that low-frequency DNA variants can be detected via low-depth multiplexed NGS after their amplification, by a median of 300-fold, using polymerase chain reaction and rationally designed 'blocker' oligonucleotides that bind to the variants. Using an 80-plex NGS panel and a sequencing depth of 250×, we detected single nucleotide polymorphisms with a VAF of 0.019% and contamination in human cell lines at a VAF as low as 0.07%. With a 16-plex NGS panel covering 145 mutations across 9 genes involved in melanoma, we detected low-VAF mutations (0.2-5%) in 7 out of the 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be notably higher than previously recognized.

Overall survival is improved when DCIS accompanies invasive breast cancer.

Invasive ductal carcinoma (IDC) often presents alone or with a co-existing ductal carcinoma in situ component (IDC + DCIS). Studies have suggested that pure IDC may exhibit different biological behavior than IDC + DCIS, but whether this translates to a difference in outcomes is unclear. Here, utilizing the National Cancer Database we identified 494,801 stage I-III breast cancer patients diagnosed with either IDC alone or IDC + DCIS. We found that IDC + DCIS was associated with significantly better overall survival (OS) compared to IDC alone (5-year OS, 89.3% vs. 85.5%, p < 0.001), and this finding persisted on multivariable Cox modeling adjusting for demographic, clinical, and treatment-related variables. The significantly superior OS observed for IDC + DCIS was limited to patients with invasive tumor size < 4 cm or with node negative disease. A greater improvement in OS was observed for tumors containing ≥25% DCIS component. We also found IDC + DCIS to be associated with lower T/N stage, low/intermediate grade, ER/PR positivity, and receipt of mastectomy. Thus, the presence of a DCIS component in patients with IDC is associated with favorable clinical characteristics and independently predicts improved OS. IDC + DCIS could be a useful prognostic factor for patients with breast cancer, particularly if treatment de-escalation is being considered for small or node negative tumors.

Early Assessment of Lung Cancer Immunotherapy Response via Circulating Tumor DNA.

Purpose: Decisions to continue or suspend therapy with immune checkpoint inhibitors are commonly guided by tumor dynamics seen on serial imaging. However, immunotherapy responses are uniquely challenging to interpret because tumors often shrink slowly or can appear transiently enlarged due to inflammation. We hypothesized that monitoring tumor cell death in real time by quantifying changes in circulating tumor DNA (ctDNA) levels could enable early assessment of immunotherapy efficacy.Experimental Design: We compared longitudinal changes in ctDNA levels with changes in radiographic tumor size and with survival outcomes in 28 patients with metastatic non-small cell lung cancer (NSCLC) receiving immune checkpoint inhibitor therapy. CtDNA was quantified by determining the allele fraction of cancer-associated somatic mutations in plasma using a multigene next-generation sequencing assay. We defined a ctDNA response as a >50% decrease in mutant allele fraction from baseline, with a second confirmatory measurement.Results: Strong agreement was observed between ctDNA response and radiographic response (Cohen's kappa, 0.753). Median time to initial response among patients who achieved responses in both categories was 24.5 days by ctDNA versus 72.5 days by imaging. Time on treatment was significantly longer for ctDNA responders versus nonresponders (median, 205.5 vs. 69 days; P < 0.001). A ctDNA response was associated with superior progression-free survival [hazard ratio (HR), 0.29; 95% CI, 0.09-0.89; P = 0.03], and superior overall survival (HR, 0.17; 95% CI, 0.05-0.62; P = 0.007).Conclusions: A drop in ctDNA level is an early marker of therapeutic efficacy and predicts prolonged survival in patients treated with immune checkpoint inhibitors for NSCLC. Clin Cancer Res; 24(8); 1872-80. ©2018 AACR.

PSA failure following definitive treatment of prostate cancer having biopsy Gleason score 7 with tertiary grade 5.

CONTEXT: In 2005, the International Society of Urologic Pathology consensus conference recommended that men with biopsy Gleason score 3 + 4 or 4 + 3 prostate cancer and tertiary pattern 5 should have their cancer classified as Gleason score 8 or 9, respectively. Yet, the management of men with Gleason score 7 vs 8 or 9 prostate cancer differs. OBJECTIVE: To compare the prognostic significance of Gleason score 7 with tertiary grade 5 vs other Gleason scores with respect to time to prostate-specific antigen (PSA) failure in men with prostate cancer. DESIGN, SETTING, AND PATIENTS: From 1989 to 2005, 2370 men with clinical tumor category 1c to 3b, node-negative, and nonmetastatic prostate cancer underwent definitive therapy with surgery or radiation therapy with or without hormonal therapy. A pathologist with expertise in genitourinary cancers assigned Gleason scores to the prostate needle biopsy specimens. Cox regression was used to assess whether a significant association existed between the presence of tertiary grade 5 in men with Gleason score 7 disease and time to recurrence compared with men with Gleason score 7 without tertiary grade 5, Gleason score 5 to 6, or 8 to 10 disease, adjusting for known prognostic factors and treatment. MAIN OUTCOME MEASURE: Time to PSA failure. RESULTS: Men with Gleason score 7 and tertiary grade 5 disease had a significantly shorter time to PSA failure than men with 7 without tertiary grade 5 (median time, 5.0 vs 6.7 years, respectively; adjusted hazard ratio (HR), 0.56; 95% confidence interval [CI], 0.32-0.97; P = .04) or score of 6 or less (median time, 15.4 years; adjusted HR, 0.24; 95% CI, 0.13-0.43; P < .001). However, a significant difference was not observed when these men were compared with men with Gleason score 8 to 10 disease (median time, 5.1 years; adjusted HR, 0.96; 95% CI, 0.54-1.71; P = .90). CONCLUSION: In this study population, men with prostate cancer having biopsy Gleason score 7 and tertiary grade 5 had a higher risk of PSA-failure when compared with men with Gleason score 7 without tertiary grade 5 and had a comparable risk with men with Gleason score 8 to 10.

Publisher Correction: Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

In the version of this Article originally published, owing to a technical error, the Life Sciences Reporting Summary was not included; this summary is now available.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don't allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

Estimation of the delivered patient dose in lung IMRT treatment based on deformable registration of 4D-CT data and Monte Carlo simulations.

The purpose of this study is to accurately estimate the difference between the planned and the delivered dose due to respiratory motion and free breathing helical CT artefacts for lung IMRT treatments, and to estimate the impact of this difference on clinical outcome. Six patients with representative tumour motion, size and position were selected for this retrospective study. For each patient, we had acquired both a free breathing helical CT and a ten-phase 4D-CT scan. A commercial treatment planning system was used to create four IMRT plans for each patient. The first two plans were based on the GTV as contoured on the free breathing helical CT set, with a GTV to PTV expansion of 1.5 cm and 2.0 cm, respectively. The third plan was based on the ITV, a composite volume formed by the union of the CTV volumes contoured on free breathing helical CT, end-of-inhale (EOI) and end-of-exhale (EOE) 4D-CT. The fourth plan was based on GTV contoured on the EOE 4D-CT. The prescribed dose was 60 Gy for all four plans. Fluence maps and beam setup parameters of the IMRT plans were used by the Monte Carlo dose calculation engine MCSIM for absolute dose calculation on both the free breathing CT and 4D-CT data. CT deformable registration between the breathing phases was performed to estimate the motion trajectory for both the tumour and healthy tissue. Then, a composite dose distribution over the whole breathing cycle was calculated as a final estimate of the delivered dose. EUD values were computed on the basis of the composite dose for all four plans. For the patient with the largest motion effect, the difference in the EUD of CTV between the planed and the delivered doses was 33, 11, 1 and 0 Gy for the first, second, third and fourth plan, respectively. The number of breathing phases required for accurate dose prediction was also investigated. With the advent of 4D-CT, deformable registration and Monte Carlo simulations, it is feasible to perform an accurate calculation of the delivered dose, and compare our delivered dose with doses estimated using prior techniques.

Ultrasensitive measurement of hotspot mutations in tumor DNA in blood using error-suppressed multiplexed deep sequencing.

Detection of cell-free tumor DNA in the blood has offered promise as a cancer biomarker, but practical clinical implementations have been impeded by the lack of a sensitive and accurate method for quantitation that is also simple, inexpensive, and readily scalable. Here we present an approach that uses next-generation sequencing to quantify the small fraction of DNA molecules that contain tumor-specific mutations within a background of normal DNA in plasma. Using layers of sequence redundancy designed to distinguish true mutations from sequencer misreads and PCR misincorporations, we achieved a detection sensitivity of approximately 1 variant in 5,000 molecules. In addition, the attachment of modular barcode tags to the DNA fragments to be sequenced facilitated the simultaneous analysis of more than 100 patient samples. As proof-of-principle, we showed the successful use of this method to follow treatment-associated changes in circulating tumor DNA levels in patients with non-small cell lung cancer. Our findings suggest that the deep sequencing approach described here may be applied to the development of a practical diagnostic test that measures tumor-derived DNA levels in blood.

Implications of respiratory motion as measured by four-dimensional computed tomography for radiation treatment planning of esophageal cancer.

PURPOSE: To evaluate the respiratory motion of primary esophageal cancers and pathologic celiac-region lymph nodes using time-resolved four-dimensional computed tomography (4D CT). METHODS AND MATERIALS: Respiration-synchronized 4D CT scans were obtained to quantify the motion of primary tumors located in the proximal, mid-, or distal thoracic esophagus, as well as any involved celiac-region lymph nodes. Respiratory motion was measured in the superior-inferior (SI), anterior-posterior (AP), and left-right (LR) directions and was analyzed for correlation with anatomic location. Recommended margin expansions were determined for both primary and nodal targets. RESULTS: Thirty patients underwent 4D CT scans at Massachusetts General Hospital for planned curative treatment of esophageal cancer. Measurements of respiratory tumor motion were obtained for 1 proximal, 4 mid-, and 25 distal esophageal tumors, as well as 12 involved celiac-region lymph nodes. The mean (SD) peak-to-peak displacements of all primary tumors in the SI, AP, and LR dimensions were 0.80 (0.45) cm, 0.28 (0.20) cm, and 0.22 (0.23) cm, respectively. Distal tumors were found to have significantly greater SI and AP motion than proximal or mid-esophageal tumors. The mean (SD) SI, AP, and LR peak-to-peak displacements of the celiac-region lymph nodes were 0.92 (0.56) cm, 0.46 (0.27) cm, and 0.19 (0.26) cm, respectively. CONCLUSIONS: Margins of 1.5 cm SI, 0.75 cm AP, and 0.75 cm LR would account for respiratory tumor motion of >95% of esophageal primary tumors in the dataset. All celiac-region lymph nodes would be adequately covered with SI, AP, and LR margins of 2.25 cm, 1.0 cm, and 0.75 cm, respectively.