Genome-wide mutational signatures in low-coverage whole genome sequencing of cell-free DNA.

Mutational signatures accumulate in somatic cells as an admixture of endogenous and exogenous processes that occur during an individual's lifetime. Since dividing cells release cell-free DNA (cfDNA) fragments into the circulation, we hypothesize that plasma cfDNA might reflect mutational signatures. Point mutations in plasma whole genome sequencing (WGS) are challenging to identify through conventional mutation calling due to low sequencing coverage and low mutant allele fractions. In this proof of concept study of plasma WGS at 0.3-1.5x coverage from 215 patients and 227 healthy individuals, we show that both pathological and physiological mutational signatures may be identified in plasma. By applying machine learning to mutation profiles, patients with stage I-IV cancer can be distinguished from healthy individuals with an Area Under the Curve of 0.96. Interrogating mutational processes in plasma may enable earlier cancer detection, and might enable the assessment of cancer risk and etiology.

PD-1 Blockade in Solid Tumors with Defects in Polymerase Epsilon.

Missense mutations in the polymerase epsilon (POLE) gene have been reported to generate proofreading defects resulting in an ultramutated genome and to sensitize tumors to checkpoint blockade immunotherapy. However, many POLE-mutated tumors do not respond to such treatment. To better understand the link between POLE mutation variants and response to immunotherapy, we prospectively assessed the efficacy of nivolumab in a multicenter clinical trial in patients bearing advanced mismatch repair-proficient POLE-mutated solid tumors. We found that only tumors harboring selective POLE pathogenic mutations in the DNA binding or catalytic site of the exonuclease domain presented high mutational burden with a specific single-base substitution signature, high T-cell infiltrates, and a high response rate to anti-PD-1 monotherapy. This study illustrates how specific DNA repair defects sensitize to immunotherapy. POLE proofreading deficiency represents a novel agnostic biomarker for response to PD-1 checkpoint blockade therapy. SIGNIFICANCE: POLE proofreading deficiency leads to high tumor mutational burden with high tumor-infiltrating lymphocytes and predicts anti-PD-1 efficacy in mismatch repair-proficient tumors. Conversely, tumors harboring POLE mutations not affecting proofreading derived no benefit from PD-1 blockade. POLE proofreading deficiency is a new tissue-agnostic biomarker for cancer immunotherapy. This article is highlighted in the In This Issue feature, p. 1397.

Considerations for Updates to ICH Q1 and Q5C Stability Guidelines: Embracing Current Technology and Risk Assessment Strategies.

In consideration of the recent ICH Quality Discussion Group (QDG) recommended revision to the ICH series of stability guidelines, the IQ Consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) Science- and Risk-based Stability Working Group conducted a comprehensive review of ICH Q1A, Q1B, Q1C, Q1D, Q1E, and Q5C to identify areas where the guidelines could be clarified, updated, and amended to reflect the potential knowledge gained from current risk-based predictive stability tools and to consider other science- and risk-based stability strategies in accordance with ICH Q8-12. The recommendations propose a holistic approach to stability understanding, utilizing historical data, prior knowledge, modeling, and a risk assessment process to expand the concept of what could be included (or would be acceptable) in the core stability data package, including type and amount of stability evidence, assignment of retest period and shelf-life for a new product, and assessment of the impact of post-approval changes.

DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity.

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

Tumor fraction-guided cell-free DNA profiling in metastatic solid tumor patients.

BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches. CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

PURPOSE: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. METHODS: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. RESULTS: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. CONCLUSION: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.

Sevoflurane formulation water content influences degradation by Lewis acids in vaporizers.

BACKGROUND: Sevoflurane is produced by several manufacturers. Currently marketed sevoflurane formulations differ in their method of synthesis, impurities, containers in which they are sold, and water content. Of the various types of chemical degradation to which sevoflurane is susceptible, the most pertinent is degradation by Lewis acids (such as metal oxides and metal halides) to hydrofluoric acid and other toxic compounds. Water inhibits such degradation. This observational study determined the degradation profile of three formulations of sevoflurane (two lower-water and one higher-water formulation) when stored in three types of vaporizers. METHODS: Lower-water sevoflurane (Eraldin, Laboratorios Richmond/Minrad, Argentina [19 ppm water] and generic sevoflurane, Baxter, US [57 ppm water]) and higher-water sevoflurane formulations (Ultane, Abbott, US [352 ppm water]) were stored in three different vaporizers (Draeger Vapor 2000, GE/Datex-Ohmeda Tec 7, Penlon Sigma Delta) under accelerated storage conditions (40 degrees C). Sevoflurane was sampled from each vaporizer immediately following filling and after 1, 2, and 3 wk, and analyzed for water content, pH, fluoride, and total degradants. RESULTS: Lower-water sevoflurane formulations stored in the Penlon Sigma Delta vaporizers contained time dependent increases in hydrofluoric acid (pH decreased as low as 3, fluoride concentration as high as 600 ppm), and total degradants (>68,000 ppm). Penlon Sigma Delta vaporizers filled with lower-water sevoflurane formulations showed substantial etching of the sight glass and metal filler shoe after 3 wk of storage. The higher-water sevoflurane formulation (Ultane, Abbott, US [352 ppm water]) contained negligible amounts of fluoride or degradants, and small decreases in pH. Sevoflurane stored in Draeger Vapor 2000 and GE/Datex-Ohmeda Tec 7 showed negligible changes in pH, fluoride concentration, and degradants. CONCLUSIONS: Lower-water sevoflurane underwent substantial degradation to hydrofluoric acid and other degradants during storage in the Penlon Sigma Delta vaporizer. Differences in water content of sevoflurane formulations and potential for degradation present a potential patient safety issue.

A Prospective Study of Circulating Tumor DNA to Guide Matched Targeted Therapy in Lung Cancers.

BACKGROUND: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) is commercially available and increasingly adopted in clinical practice despite a paucity of prospective data to support its use. METHODS: Patients with advanced lung cancers who had no known oncogenic driver or developed resistance to current targeted therapy (n = 210) underwent plasma NGS, targeting 21 genes. A subset of patients had concurrent tissue NGS testing using a 468-gene panel (n = 106). Oncogenic driver detection, test turnaround time (TAT), concordance, and treatment response guided by plasma NGS were measured. All statistical tests were two-sided. RESULTS: Somatic mutations were detected in 64.3% (135/210) of patients. ctDNA detection was lower in patients who were on systemic therapy at the time of plasma collection compared with those who were not (30/70, 42.9% vs 105/140, 75.0%; OR = 0.26, 95% CI = 0.1 to 0.5, P < .001). The median TAT of plasma NGS was shorter than tissue NGS (9 vs 20 days; P < .001). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 56.6% (60/106, 95% CI = 46.6% to 66.2%). Among patients who tested plasma NGS positive, 89.6% (60/67; 95% CI = 79.7% to 95.7%) were also concordant on tissue NGS and 60.6% (60/99; 95% CI = 50.3% to 70.3%) vice versa. Patients who tested plasma NGS positive for oncogenic drivers had tissue NGS concordance of 96.1% (49/51, 95% CI = 86.5% to 99.5%), and directly led to matched targeted therapy in 21.9% (46/210) with clinical response. CONCLUSIONS: Plasma ctDNA NGS detected a variety of oncogenic drivers with a shorter TAT compared with tissue NGS and matched patients to targeted therapy with clinical response. Positive findings on plasma NGS were highly concordant with tissue NGS and can guide immediate therapy; however, a negative finding in plasma requires further testing. Our findings support the potential incorporation of plasma NGS into practice guidelines.

Liquid biopsy for ctDNA to revolutionize the care of patients with early stage lung cancers.

The current standard of adjuvant therapies for patients with early stage non-small-cell lung cancers largely depends on the stage of disease. Liquid biopsy for circulating tumor DNA (ctDNA) has the potential to detect minimal residual disease, depict genomic evolution, guide precision medicine to individual patients and revolutionize the management of early stage lung cancers. In light of the seminal work published by Abbosh and colleagues, we discuss the potential paradigm changing clinical implications of ctDNA, the biological and technological challenges to consider, and the future of ctDNA driven therapeutic studies.

Polyanhydride implant for antibiotic delivery--from the bench to the clinic.

A polyanhydride implant (Septacin) containing gentamicin sulfate was developed for sustained local delivery of the drug to the site of infection in the treatment of osteomyelitis. Laboratory-scale injection molding equipment was utilized to fabricate the implant for in vitro characterization. Molding conditions were optimized to produce implants with a skin-core structure which was found to be critical in preventing the initial cracking of the implant during in vitro drug release test in water. A manufacturing process consisting of twin-screw extrusion, pelletizing, and injection molding was developed. Polymer-drug pellets were characterized with respect to copolymer molecular weight and drug content uniformity. The implants were terminally sterilized by gamma-radiation which was found to cause increase in copolymer molecular weight as a result of polymer chain extension. The stability of Septacin was evaluated as a function of storage temperature and time. A marked decline in copolymer molecular weight occurred in samples stored above freezing temperatures and significantly slower drug-release profiles were also exhibited by these samples. In vivo drug release from Septacin in rats showed that the gentamicin plasma levels were extremely low, indicating the low systemic exposure to gentamicin. Furthermore, Septacin samples have demonstrated efficacy in the rat skin-abscess and horse-joint infection models. Results from a human in vivo study also showed high local drug concentrations at implantation sites while systemic exposure to the drug was minimal.

Effect of gamma-radiation on a polyanhydride implant containing gentamicin sulfate.

Septacin, a polyanhydride implant containing gentamicin sulfate, was sterilized by gamma-radiation. Its copolymer molecular weight (M(w) by GPC) was increased after this radiation. No cross-linking was shown in the radiated samples as no gel content was found by the filtration method. The chemical structure as detected by 1H NMR for non-radiated and radiated samples was comparable. For samples radiated at higher dose levels (70-100 kGy), the IR spectra showed that the intensity of absorbance attributable to the C-H stretching vibration (at 2852 and 2927 cm(-1)) was attenuated, indicating free-radical formation or loss of hydrogen atoms from C-H bonds. However, the mass spectra for the gamma-radiated and the non-radiated controls after they were completely depolymerized in methylene chloride were virtually identical. Therefore, it could be concluded that the increase in copolymer molecular weight for radiated Septacin was a result of chain extension in the copolymer backbone during radiation. In addition, wide-angle X-ray diffraction and polarizing light microscopy (PLM) revealed a change in the physical structure of the radiated copolymer. There was an increase in crystallinity of the copolymer with increasing radiation doses; the greatest increase in crystallinity occurred at the dose range of 70-80 kGy, which was also shown to result in the greatest molecular-weight increase. The crystalline morphology of the samples as detected by PLM was not altered by gamma-radiation, regardless of the dose levels.

The development of an injection-molding process for a polyanhydride implant containing gentamicin sulfate.

A production-scale manufacturing process has been developed for polyanhydride/gentamicin sulfate implants for the treatment of osteomyelitis. Gentamicin sulfate was first dried to an acceptable moisture level by using a tumble vacuum dryer. Dried gentamicin sulfate powder and polyanhydride granules were separately fed into the twin-screw extruder at a pre-determined metering rate using a gravimetric feeding device. The extruded molten mixture was solidified to form strands which were subsequently cut into pellets by using a pelletizer. The pellets were characterized with respect to copolymer molecular weight and drug content uniformity. The pellets were later fed into production-scale injection-molding equipment for implant fabrication. The injection-molding cycle was developed and evaluated in terms of cycle reproducibility. Implants were tested and shown to yield an oriented skin-core structure exhibiting a desirable in-vitro drug release profile.

Targeted cancer therapy by immunoconjugated gold-gold sulfide nanoparticles using Protein G as a cofactor.

Gold-gold sulfide nanoparticles (GGS-NPs) fabricated from chloroauric acid and sodium thiosulfate show unique near infrared (NIR) absorption that renders them as a promising candidate for photothermal cancer therapy. To improve targeting efficiency, we developed a versatile method to allow ordered immunoconjugation of antibodies on the surfaces of these nanoparticles via a PEGylated recombinant Protein G (ProG). The PEGylated ProG was prepared with orthopyridyldisulfide-polyethylene glycol-succinimidyl valerate, average MW 2000 (OPSS-PEG-SVA), to first allow the self-assembly of ProG on the nanoparticles, subsequently antibodies were added to this construct to enable active targeting. The bioconjugated GGS-NPs were characterized by TEM, NIR-spectra, dynamic light scattering and modified immunoassay. In in vitro studies, the ProG-conjugated GGS-NPs with bound mouse anti c-erbB-2 (HER-2) immunoglobulin G (IgG) successfully targeted the HER-2 overexpressing breast cancer cell, SK-BR-3. Extensive cell death was observed for the targeted SK-BR-3 line at a low laser power of 540 J (3 W cm(-2) for 3 min) while the control breast cancer cell (low expressing HER-2), HTB-22 survived. Using PEGylated ProG as a cofactor for immobilization of antibodies offers a promising strategy to functionalize various IgGs on nanoparticles for engineering their biomedical applications in cancer therapeutics.

Tunability and stability of gold nanoparticles obtained from chloroauric Acid and sodium thiosulfate reaction.

In the quest for producing an effective clinically relevant therapeutic agent, scalability, repeatability, and stability are paramount. In this paper, gold nanoparticles (GNPs) with precisely controlled near infrared (NIR) absorption are synthesized by a single step reaction of HAuCl4 and Na2S2O3, without assistance of additional templates, capping reagents or seeds. The anisotropy in the shape of gold nanoparticles offers high NIR absorption making it therapeutically relevant. The synthesized products consist of GNPs with different shape and size, including small spherical colloid gold particles and non-spherical gold crystals. The NIR absorption wavelengths and particle size increase with increasing molar ratio of HAuCl4/Na2S2O3. Non-spherical gold particles can be further purified and separated by centrifugation to improve the NIR absorbing fraction of particles. In-depth studies reveal that GNPs with good structural and optical stability only form in a certain range of the HAuCl4/Na2S2O3 molar ratio, whereas higher molar ratios result in unstable GNPs, which lose their NIR absorption peak due to decomposition and reassembly via Ostwald ripening. Tuning the optical absorption of the gold nanoparticles in the NIR regime via a robust and repeatable method will improve many applications requiring large quantities of desired NIR absorbing nanoparticles.

Heat-induced formulation inhomogeneity of a three-component suspension.

A suspension formulation containing sarafloxacin HCl, triamcinolone acetonide, and clotrimazole was developed for the treatment of otitis externa in dogs. The potency for the three active ingredients in this suspension was monitored at 25 degrees C and 40 degrees C for up to 3 months. The potencies of triamcinolone and clotrimazole were found unchanged, but the potency of sarafloxacin HCl in the samples stored at 40 degrees C for 1 month varied significantly between samples. However, assay inconsistency for sarafloxacin HCl was not seen in samples stored at 25 degrees C. Under an optical microscope, large crystals were found in the 40 degrees C stability samples but not in the 25 degrees C samples. The large crystals in 40 degrees C samples were identified as sarafloxacin by high-performance liquid chromatography (HPLC). This finding suggests that crystal growth of sarafloxacin took place at 40 degrees C during storage, leading to the formation of larger crystals and the consequent sampling nonuniformity and assay inconsistency. The solid-state properties of these crystals were further evaluated using hot-stage microscopy and Fourier transform infrared (FTIR) analysis. The results indicate that the crystal growth of sarafloxacin was most likely attributed to a change in the hydration form of sarafloxacin.