Single duplex DNA sequencing with CODEC detects mutations with high sensitivity.

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Circulating tumor DNA association with residual cancer burden after neoadjuvant chemotherapy in triple-negative breast cancer in TBCRC 030.

Purpose: To examine circulating tumor DNA (ctDNA) and its association with residual cancer burden (RCB) using an ultrasensitive assay in patients with triple-negative breast cancer (TNBC) receiving neoadjuvant chemotherapy (NAT). Patients and Methods: We identified responders (RCB-0/1) and matched non-responders (RCB-2/3) from the phase II TBCRC 030 prospective study of neoadjuvant paclitaxel vs. cisplatin in TNBC. We collected plasma samples at baseline, three weeks, and twelve weeks (end of therapy). We created personalized ctDNA assays utilizing MAESTRO mutation enrichment sequencing. We explored associations between ctDNA and RCB status and disease recurrence. Results: Of 139 patients, 68 had complete samples and no additional NAT. Twenty-two were responders and 19 of those had sufficient tissue for whole-genome sequencing. We identified an additional 19 non-responders for a matched case-control analysis of 38 patients using a MAESTRO ctDNA assay tracking 319-1000 variants (median 1000) to 114 plasma samples from 3 timepoints. Overall, ctDNA positivity was 100% at baseline, 79% at week 3, and 55% at week 12. Median tumor fraction (TFx) was 3.7 × 10 -4 (range: 7.9 × 10 -7 to 4.9 × 10 -1 ). TFx decreased 285-fold from baseline to week 3 in responders and 24-fold in non-responders. Week 12 ctDNA clearance correlated with RCB: clearance was observed in 10/11 patients with RCB-0, 3/8 with RCB-1, 4/15 with RCB-2, and 0/4 with RCB-3. Among 6 patients with known recurrence five had persistent ctDNA at week 12. Conclusion: NAT for TNBC reduced ctDNA TFx by 285-fold in responders and 24-fold in non-responders. In 58% (22/38) of patients, ctDNA TFx dropped below the detection level of a commercially available test, emphasizing the need for sensitive tests. Additional studies will determine if ctDNA-guided approaches can improve outcomes.

Enzymatic Methods for Mutation Detection in Cancer Samples and Liquid Biopsies.

Low-level tumor somatic DNA mutations in tissue and liquid biopsies obtained from cancer patients can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up, or early cancer detection. Unless detected, such low-frequency DNA alterations can misinform patient management decisions or become missed opportunities for personalized medicine. Next-generation sequencing technologies and digital-PCR can resolve low-level mutations but require access to specialized instrumentation, time, and resources. Enzymatic-based approaches to detection of low-level mutations provide a simple, straightforward, and affordable alternative to enrich and detect such alterations and is broadly available to low-resource laboratory settings. This review summarizes the traditional uses of enzymatic mutation detection and describes the latest exciting developments, potential, and applications with specific reference to the field of liquid biopsy in cancer.

Combined clinical and research training in medical physics in a multi-institutional setting: 13-year experience of Harvard Medical Physics Residency Program.

PURPOSE: This manuscript describes the structure, management and outcomes of a multi-institutional clinical and research medical physics residency program (Harvard Medical Physics Residency Program, or HMPRP) to provide potentially useful information to the centers considering a multi-institutional approach for their training programs. METHODS: Data from the program documents and public records was used to describe HMPRP and obtain statistics about participating faculty, enrolled residents, and graduates. Challenges associated with forming and managing a multi-institutional program and developed solutions for effective coordination between several clinical centers are described. RESULTS: HMPRP was formed in 2009 and was accredited by the Commission on Accreditation of Medical Physics Education Programs (CAMPEP) in 2011. It is a 3-year therapy program, with a dedicated year of research and the 2 years of clinical training at three academic hospitals. A CAMPEP-accredited Certificate Program is embedded in HMPRP to allow enrolled residents to complete a formal didactic training in medical physics if necessary. The clinical training covers the material required by CAMPEP. In addition, training in protons, CyberKnife, MR-linac, and at network locations is included. The clinical training and academic record of the residents is outstanding. All graduates have found employment within clinical medical physics, mostly at large academic centers and graduates had a 100% pass rate at the oral American Board of Radiology exams. On average, three manuscripts per resident are published during residency, and multiple abstracts are presented at conferences. CONCLUSIONS: A multi-institutional medical physics residency program can be successfully formed and managed. With a collaborative administrative structure, the program creates an environment for high-quality clinical training of the residents and high productivity in research. The main advantage of such program is access to a wide variety of resources. The main challenge is creating a structure for efficient management of multiple resources at different locations. This report may provide valuable information to centers considering starting a multi-institutional residency program.

Pre-PCR Mutation-Enrichment Methods for Liquid Biopsy Applications.

Liquid biopsy is having a remarkable impact on healthcare- and disease-management in the context of personalized medicine. Circulating free DNA (cfDNA) is one of the most instructive liquid-biopsy-based biomarkers and harbors valuable information for diagnostic, predictive, and prognostic purposes. When it comes to cancer, circulating DNA from the tumor (ctDNA) has a wide range of applications, from early cancer detection to the early detection of relapse or drug resistance, and the tracking of the dynamic genomic make-up of tumor cells. However, the detection of ctDNA remains technically challenging, due, in part, to the low frequency of ctDNA among excessive circulating cfDNA originating from normal tissues. During the past three decades, mutation-enrichment methods have emerged to boost sensitivity and enable facile detection of low-level mutations. Although most developed techniques apply mutation enrichment during or following initial PCR, there are a few techniques that allow mutation selection prior to PCR, which provides advantages. Pre-PCR enrichment techniques can be directly applied to genomic DNA and diminish the influence of PCR errors that can take place during amplification. Moreover, they have the capability for high multiplexity and can be followed by established mutation detection and enrichment technologies without changes to their established procedures. The first approaches for pre-PCR enrichment were developed by employing restriction endonucleases directly on genomic DNA in the early 1990s. However, newly developed pre-PCR enrichment methods provide higher sensitivity and versatility. This review describes the available pre-PCR enrichment methods and focuses on the most recently developed techniques (NaME-PrO, UVME, and DEASH/MAESTRO), emphasizing their applications in liquid biopsies.

Recent Developments in Mutation Enrichment and Detection Technologies.

BACKGROUND: Presence of excess unaltered, wild-type DNA (wtDNA) providing information of little clinical value may often mask low-level mutations containing important diagnostic or therapeutic clues. This is a recurring hurdle in biotechnology and medicine, including cancer, prenatal diagnosis, infectious diseases, and organ transplantation. Mutation enrichment techniques that allow reduction of unwanted DNA to enable the detection of low-level mutations have emerged since the early 1990s. They are continuously being refined and updated with new technologies. The burgeoning interest in liquid biopsies for residual cancer monitoring, detection of resistance to therapy, and early cancer detection has driven an expanded interest in new and improved methodologies for practical and effective mutation enrichment and detection of low-level mutations of clinical relevance. CONTENT: Newly developed mutation enrichment technologies are described and grouped according to the main principle of operation, PCR-blocking technologies, enzymatic methods, and physicochemical approaches. Special emphasis is given to technologies enabling pre-PCR blockage of wtDNA to bypass PCR errors [nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO) and UV-mediated cross-linking minor allele enrichment (UVME)] or providing high multiplexity followed by next-generation sequencing [Minor allele enriched sequencing through recognition oligonucleotides (MAESTRO)]. SUMMARY: This review summarizes technological developments in rare mutation enrichment over the last 12 years, complementing pre-2010 reviews on this topic. The expanding field of liquid biopsy calls for improved limits of detection (LOD) and highly parallel applications, along with the traditional requirements for accuracy, speed, and cost-effectiveness. The current technologies are reviewed with regards to these new requirements.

Nuclease-Assisted, Multiplexed Minor-Allele Enrichment: Application in Liquid Biopsy of Cancer.

The use of next-generation sequencing (NGS) to profile genomic variation of individual cancer species is revolutionizing the practice of clinical oncology. In liquid biopsy of cancer, sequencing of circulating-free DNA (cfDNA) is gradually applied to all stages of cancer diagnosis and treatment, serving as complement or replacement of tissue biopsies. However, analysis of cfDNA obtained from blood draws still faces technical obstacles due in part to an excess of wild-type DNA originating from normal tissues and hematopoietic cells. The resulting low-level mutation abundance often falls below routine NGS detection sensitivity and limits reliable mutation identification that meets clinical sensitivity and specificity standards. Despite sample preparation advances that reduce sequencing error rates via use of unique molecular identifiers (molecular barcodes) and error-suppression algorithms, excessive amounts of sequencing are still required to detect mutations at allelic frequency levels below 1%. This requirement reduces throughput and increases cost.In this chapter, we describe a sensitive multiplex mutation detection method that enriches mutation-containing DNA during sample preparation, prior to sequencing, thereby increasing signal-to-noise ratios and providing low-level mutation detection without excessive sequencing depth. We couple targeted next-generation sequencing with wild-type DNA removal using Nuclease-assisted Minor-allele Enrichment using Probe Overlap, NaME-PrO, a recently developed method to eliminate wild-type sequences from multiple targets simultaneously. A step by step guide to library preparation and data analysis are provided as well as some precautions during the sample handling.

Boosting the Abscopal Effect Using Immunogenic Biomaterials With Varying Radiation Therapy Field Sizes.

PURPOSE: Persistent immunosuppression in the tumor microenvironment is a major limitation to boosting the abscopal effect, whereby radiation therapy at 1 site can lead to regression of tumors at distant sites. Here, we investigate the use of radiation and immunogenic biomaterials (IBM) targeting only the gross tumor volume/subvolume for boosting the abscopal effect in immunologically cold tumors. METHODS AND MATERIALS: To evaluate the abscopal effect, 2 syngeneic contralateral tumors were implanted in each mouse, where only 1 tumor was treated. IBM was administered to the treated tumor with 1 fraction of radiation and results were compared, including as a function of different radiation therapy field sizes. The IBM was designed similar to fiducial markers using immunogenic polymer components loaded with anti-CD40 agonist. Tumor volumes of both treated and untreated tumors were measured over time, along with survival and corresponding immune cell responses. RESULTS: Results showed that radiation with IBM administered to the gross tumor subvolume can effectively boost abscopal responses in both pancreatic and prostate cancers, significantly increasing survival (P < .0001 and P < .001, respectively). Results also showed equal or superior abscopal responses when using field sizes smaller than the gross tumor volume compared with irradiating the whole tumor volume. These results were buttressed by observation of higher infiltration of cytotoxic CD8+ T-lymphocytes in the treated tumors (P < .0001) and untreated tumors (P < .0001) for prostate cancer. Significantly higher infiltration was also observed in treated tumors (P < .0001) and untreated tumors P < .01) for pancreatic cancer. Moreover, the immune responses were accompanied by a positive shift of proinflammatory cytokines in both prostate and pancreatic tumors. CONCLUSIONS: The approach targeting gross tumor subvolumes with radiation and IBM offers opportunity for boosting the abscopal effect while significantly minimizing healthy tissue toxicity. This approach proffers a radioimmunotherapy dose-painting strategy that can be developed for overcoming current barriers of immunosuppression especially for immunologically cold tumors.

Mutation enrichment in human DNA samples via UV-mediated cross-linking.

Detection of low-level DNA mutations can reveal recurrent, hotspot genetic changes of clinical relevance to cancer, prenatal diagnostics, organ transplantation or infectious diseases. However, the high excess of wild-type (WT) alleles, which are concurrently present, often hinders identification of salient genetic changes. Here, we introduce UV-mediated cross-linking minor allele enrichment (UVME), a novel approach that incorporates ultraviolet irradiation (∼365 nm UV) DNA cross-linking either before or during PCR amplification. Oligonucleotide probes matching the WT target sequence and incorporating a UV-sensitive 3-cyanovinylcarbazole nucleoside modification are employed for cross-linking WT DNA. Mismatches formed with mutated alleles reduce DNA binding and UV-mediated cross-linking and favor mutated DNA amplification. UV can be applied before PCR and/or at any stage during PCR to selectively block WT DNA amplification and enable identification of traces of mutated alleles. This enables a single-tube PCR reaction directly from genomic DNA combining optimal pre-amplification of mutated alleles, which then switches to UV-mediated mutation enrichment-based DNA target amplification. UVME cross-linking enables enrichment of mutated KRAS and p53 alleles, which can be screened directly via Sanger sequencing, high-resolution melting, TaqMan genotyping or digital PCR, resulting in the detection of mutation allelic frequencies of 0.001-0.1% depending on the endpoint detection method. UV-mediated mutation enrichment provides new potential for mutation enrichment in diverse clinical samples.

Duplex-Repair enables highly accurate sequencing, despite DNA damage.

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via 'End Repair/dA-Tailing,' may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior 'duplex base pairs' from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles' heel in sequencing and offers a solution to restore high accuracy.

A bi-institutional multi-disciplinary failure mode and effects analysis (FMEA) for a Co-60 based total body irradiation technique.

BACKGROUND: We aim to assess the risks associated with total body irradiation (TBI) delivered using a commercial dedicated Co-60 irradiator, and to evaluate inter-institutional and inter-professional variations in the estimation of these risks. METHODS: A failure mode and effects analysis (FMEA) was generated using guidance from the AAPM TG-100 report for quantitative estimation of prospective risk metrics. Thirteen radiation oncology professionals from two institutions rated possible failure modes (FMs) for occurrence (O), severity (S), and detectability (D) indices to generate a risk priority number (RPN). The FMs were ranked by descending RPN value. Absolute gross differences (AGD) in resulting RPN values and Jaccard Index (JI; for the top 20 FMs) were calculated. The results were compared between professions and institutions. RESULTS: A total of 87 potential FMs (57, 15, 10, 3, and 2 for treatment, quality assurance, planning, simulation, and logistics respectively) were identified and ranked, with individual RPN ranging between 1-420 and mean RPN values ranging between 6 and 74. The two institutions shared 6 of their respective top 20 FMs. For various institutional and professional comparison pairs, the number of common FMs in the top 20 FMs ranged from 6 to 13, with JI values of 18-48%. For the top 20 FMs, the trend in inter-professional variability was institution-specific. The mean AGD values ranged between 12.5 and 74.5 for various comparison pairs. AGD values differed the most for medical physicists (MPs) in comparison to other specialties i.e. radiation oncologists (ROs) and radiation therapists (RTs) [MPs-vs-ROs: 36.3 (standard deviation SD = 34.1); MPs-vs-RTs: 41.2 (SD = 37.9); ROs-vs-RTs: 12.5 (SD = 10.8)]. Trends in inter-professional AGD values were similar for both institutions. CONCLUSION: This inter-institutional comparison provides prospective risk analysis for a new treatment delivery unit and illustrates the institution-specific nature of FM prioritization, primarily due to operational differences. Despite being subjective in nature, the FMEA is a valuable tool to ensure the identification of the most significant risks, particularly when implementing a novel treatment modality. The creation of a bi-institutional, multidisciplinary FMEA for this unique TBI technique has not only helped identify potential risks but also served as an opportunity to evaluate clinical and safety practices from the perspective of both multiple professional roles and different institutions.

Sensitive detection of microsatellite instability in tissues and liquid biopsies: Recent developments and updates.

Microsatellite instability (MSI), a phenotype displayed as deletions/insertions of repetitive genomic sequences, has drawn great attention due to its application in cancer including diagnosis, prognosis and immunotherapy response prediction. Several methods have been developed for the detection of MSI, facilitating the MSI classification of cancer patients. In view of recent interest in minimally-invasive detection of MSI via liquid biopsy samples, which requires methods with high sensitivity to identify small fractions of altered DNA in the presence of large amount of wild type copies, sensitive MSI detection approaches are emerging. Here we review the available MSI detection methods and their detection limits and focus on recently developed next-generation-sequencing based approaches and bioinformatics algorithms available for MSI analysis in various cancer types.

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies.

A considerable number of estrogen receptor-positive breast cancer (ER+ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER+ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER+ BrCa primary tumour tissues, 13 non-cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM-positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation-allelic-frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow-up period in the same patient. In the EpCAM-isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts.

NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR.

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80-150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05-1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.

Noninvasive imaging of tumor hypoxia after nanoparticle-mediated tumor vascular disruption.

We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, including tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disruption. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nanoparticles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model.

Author Correction: Image-guided radiotherapy platform using single nodule conditional lung cancer mouse models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.