Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium.

BACKGROUND: Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration. PATIENTS AND METHODS: Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases. RESULTS: In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification. CONCLUSIONS: Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.

Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC).

Background: Neoadjuvant anti-PD-1 may improve outcomes for patients with resectable NSCLC and provides a critical window for examining pathologic features associated with response. Resections showing major pathologic response to neoadjuvant therapy, defined as ≤10% residual viable tumor (RVT), may predict improved long-term patient outcome. However, %RVT calculations were developed in the context of chemotherapy (%cRVT). An immune-related %RVT (%irRVT) has yet to be developed. Patients and methods: The first trial of neoadjuvant anti-PD-1 (nivolumab, NCT02259621) was just reported. We analyzed hematoxylin and eosin-stained slides from the post-treatment resection specimens of the 20 patients with non-small-cell lung carcinoma who underwent definitive surgery. Pretreatment tumor biopsies and preresection radiographic 'tumor' measurements were also assessed. Results: We found that the regression bed (the area of immune-mediated tumor clearance) accounts for the previously noted discrepancy between CT imaging and pathologic assessment of residual tumor. The regression bed is characterized by (i) immune activation-dense tumor infiltrating lymphocytes with macrophages and tertiary lymphoid structures; (ii) massive tumor cell death-cholesterol clefts; and (iii) tissue repair-neovascularization and proliferative fibrosis (each feature enriched in major pathologic responders versus nonresponders, P < 0.05). This distinct constellation of histologic findings was not identified in any pretreatment specimens. Histopathologic features of the regression bed were used to develop 'Immune-Related Pathologic Response Criteria' (irPRC), and these criteria were shown to be reproducible amongst pathologists. Specifically, %irRVT had improved interobserver consistency compared with %cRVT [median per-case %RVT variability 5% (0%-29%) versus 10% (0%-58%), P = 0.007] and a twofold decrease in median standard deviation across pathologists within a sample (4.6 versus 2.2, P = 0.002). Conclusions: irPRC may be used to standardize pathologic assessment of immunotherapeutic efficacy. Long-term follow-up is needed to determine irPRC reliability as a surrogate for recurrence-free and overall survival.

Risk of malignancy in resected cystic tumors of the pancreas < or =3 cm in size: is it safe to observe asymptomatic patients? A multi-institutional report.

Recent international consensus guidelines propose that cystic pancreatic tumors less than 3 cm in size in asymptomatic patients with no radiographic features concerning for malignancy are safe to observe; however, there is little published data to support this recommendation. The purpose of this study was to determine the prevalence of malignancy in this group of patients using pancreatic resection databases from five high-volume pancreatic centers to assess the appropriateness of these guidelines. All pancreatic resections performed for cystic neoplasms < or =3 cm in size were evaluated over the time period of 1998-2006. One hundred sixty-six cases were identified, and the clinical, radiographic, and pathological data were reviewed. The correlation with age, gender, and symptoms (abdominal pain, nausea and vomiting, jaundice, presence of pancreatitis, unexplained weight loss, and anorexia), radiographic features suggestive of malignancy by either computed tomography, magnetic resonance imaging, or endoscopic ultrasound (presence of solid component, lymphadenopathy, or dilated main pancreatic duct or common bile duct), and the presence of malignancy was assessed using univariate and multivariate analysis. Among the 166 pancreatic resections for cystic pancreatic tumors < or =3 cm, 135 cases were benign [38 serous cystadenomas, 35 mucinous cystic neoplasms, 60 intraductal papillary mucinous neoplasms (IPMN), 1 cystic papillary tumor, and 1 cystic islet cell tumor], whereas 31 cases were malignant (14 mucinous cystic adenocarcinomas and 13 invasive carcinomas and 4 in situ carcinomas arising in the setting of IPMN). A greater incidence of cystic neoplasms was seen in female patients (99/166, 60%). Gender was a predictor of malignant pathology, with male patients having a higher incidence of malignancy (19/67, 28%) compared to female patients (12/99, 12%; p < 0.02). Older age was associated with malignancy (mean age 67 years in patients with malignant disease vs 62 years in patients with benign lesions (p < 0.05). A majority of the patients with malignancy were symptomatic (28/31, 90%). Symptoms that correlated with malignancy included jaundice (p < 0.001), weight loss (p < 0.003), and anorexia (p < 0.05). Radiographic features that correlated with malignancy were presence of a solid component (p < 0.0001), main pancreatic duct dilation (p = 0.002), common bile duct dilation (p < 0.001), and lymphadenopathy (p < 0.002). Twenty-seven of 31(87%) patients with malignant lesions had at least one radiographic feature concerning for malignancy. Forty-five patients (27%) were identified as having asymptomatic cystic neoplasms. All but three (6.6%) of the patients in this group had benign disease. Of the patients that had no symptoms and no radiographic features, 1 out of 30 (3.3%) had malignancy (carcinoma in situ arising in a side branch IPMN). Malignancy in cystic neoplasms < or =3 cm in size was associated with older age, male gender, presence of symptoms (jaundice, weight loss, and anorexia), and presence of concerning radiographic features (solid component, main pancreatic duct dilation, common bile duct dilation, and lymphadenopathy). Among asymptomatic patients that displayed no discernable radiographic features suggestive of malignancy who underwent resection, the incidence of occult malignancy was 3.3%. This study suggests that a group of patients with small cystic pancreatic neoplasms who have low risk of malignancy can be identified, and selective resection of these lesions may be appropriate.

Effects of functional group interactions on the gas-phase methylation and dissociation of acids and esters.

Functional group interactions have been observed to affect gas-phase ion-molecule chemistry in a quadrupole ion trap mass spectrometer. Gas-phase methylation and collisionactivated dissociation reactions of a series of related acids and esters allows an evaluation of the structural factors that influence reactivity and functional group interactions of these compounds. Examination of the [M+H](+) or [M+15](+) product ions by collision-activated dissociation has provided insight into the conformations from which diacids and diesters undergo electrophilic addition. Collision-activated dissociation has provided not only more detailed information on the structures of the ions, but also the data necessary for confident mechanistic interpretation. Labeling studies were done to probe fragmentation pathways. Upon activation of the [M+CD3](+) products of dimethyl maleate and dimethyl succinate, formed from reaction of the neutrals with CD3OCD 2 (+) ions, a rapid interfunctional group methyl transfer causes scrambling of the methyls prior to elimination of dimethyl ether or methanol. The [M+15](+) ions of dimethyl maleate are believed to lose dimethyl ether through a rate-determining 1,6-methyl transfer, whereas the [M+15](+) ions of dimethyl succinate eliminate methanol through a rate-determining 1,5-proton transfer.

A non-radioactive fluorescent method for measuring protein kinase C activity.

The purpose of this study is to report the development of a non-radioactive fluorescent peptide assay for measuring protein kinase C activity (PKC). The assay is based on a glycogen synthase derived fluorescent peptide that is phosphorylated by PKC. Phosphorylation causes the peptide to migrate toward the anode while the non-phosphorylated peptide migrates toward the cathode during agarose gel electrophoresis. Quantitation of PKC activity can be accomplished by excision of the appropriate bands and measuring their relative fluorescence. Using this assay, PKC activity was measured in whole cell homogenates from cultured renal mesangial cells. The enzyme(s)-substrate system followed Michaelis-Menten kinetics under limited conditions and, therefore, Lineweaver-Burk plots were used to obtain Michaelis constant and maximum velocity values. An apparent KM value of 40 microM was obtained for the fluorescent peptide substrate with a control Vmax value of 300 pmol/min. Addition of phorbol 12-myristate 13-acetate increased Vmax to 380 pmol/min.

Permian polar forests: deciduousness and environmental variation.

Forests are expected to expand into northern polar latitudes in the next century. However, the impact of forests at high latitudes on climate and terrestrial biogeochemical cycling is poorly understood because such forests cannot be studied in the modern. This study presents forestry and geochemical analyses of three in situ fossil forests from Late Permian strata of Antarctica, which grew at polar latitudes. Stem size measurements and stump spacing measurements indicate significant differences in forest density and canopy structure that are related to the local depositional setting. For forests closest to fluvial systems, tree density appears to decrease as the forests mature, which is the opposite trend of self-thinning observed in modern forests. We speculate that a combination of tree mortality and high disturbance created low-density mature forests without understory vegetation near Late Permian river systems. Stable carbon isotopes measured from permineralized wood in these forests demonstrate two important points: (i) recently developed techniques of high-resolution carbon isotope studies of wood and mummified wood can be applied to permineralized wood, for which much of the original organic matter has been lost and (ii) that the fossil trees maintained a deciduous habit at polar latitudes during the Late Permian. The combination of paleobotanical, sedimentologic, and paleoforestry techniques provides an unrivaled examination of the function of polar forests in deep time; and the carbon isotope geochemistry supplements this work with subannual records of carbon fixation that allows for the quantitative analysis of deciduous versus evergreen habits and environmental parameters, for example, relative humidity.

Use of borinium ions as probes of steric effects in gas-phase ion-molecule complexes.

The relative free energies of binding of the dimethoxy borinium ion to several substituted pyridines were determined using the equilibrium method and were found to correlate well with relative gas-phase basicities except in the case in which different sterics were present near the site of cation attachment. Dimethoxy borinium ion binds to 3,5-dimethylpyridine and 3,4-dimethylpyridine 2.5+0.4 and 3.5+0.3 kcal/mole more strongly than it does to 3-methylpyridine. 2,4-Dimethylpyridine shows a 2.0+0.4 kcal/mole preference over 2-methylpyridine for dimethoxy borinium ion attachment. This difference is in agreement with the difference in gas-phase basicities of these two compounds, suggesting the borinium ion binds to the same position as the proton. 2,3-Dimethylpyridine deviated from this trend, with its borinium ion complex being 1.5+0.4 kcal/mole less stable than that of 3-methylpyridine.

Realities of high-throughput liquid chromatography/mass spectrometry purification of large combinatorial libraries: a report on overall sample throughput using parallel purification.

We report on the development of a validated, streamlined high-throughput process for the purification of parallel-synthesis-derived combinatorial libraries. The steps involved in this library purification process include dissolution of dry films of crude synthetic material, dual-column LC/MS purification, dual-column postpurification analysis, quantitation, reformatting, and submission of pure compounds for registration. Although the purification and postpurification analysis times decreased essentially linearly as a function of the number of HPLC columns employed, it was not possible to decrease the total purification process time linearly as a function of the number of columns employed in the system. This was due primarily to the fact that numerous steps in the total purification process are independent of sample analysis and purification (e.g., evaporation, reconstitution, and reformatting, etc.). Additionally, experiments were also performed to assess whether separate gradient pumps were necessary for each channel of this two-channel LC/MS or if acceptable results could be reliably obtained by splitting the flow from one set of gradient pumps between two HPLC columns. On the basis of the parallel, two-column LC/MS system employed in this work, throughput estimates were extrapolated to more massively parallel systems (e.g., four-channel LC/MS).

Oxidation inhibits substrate proteolysis by calpain I but not autolysis.

In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80- and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 microM, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 microM. In addition, autolytic conversion of either the 80- or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4- methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.