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.

Long-term treatment of antiphospholipid syndrome-associated cerebral arterial thromboses with intravenous immunoglobulin: a case report.

We report a now 13-year-old male with trisomy 21, hypothyroidism, and insulin-dependent diabetes who developed acute hemiplegia due to the antiphospholipid antibody syndrome (APS) at age four. The risks of long-term anticoagulation were initially considered to be high; hence, he was treated with monthly infusions of intravenous immunoglobulin (IVIG) at 2 g/kg for 2 years and then every other month for 7 years. Antiphospholipid antibodies were no longer detectable within 6 months and have continued to be negative. There was no clinical deterioration or further changes on magnetic resonance arteriography over 7 years. IVIG may be an alternative therapeutic choice for children with APS who are not candidates for conventional anticoagulation.

Classification of diverse organic compounds that induce chromosomal aberrations in Chinese hamster cells.

A data set of 297 diverse organic compounds that cause varying degrees of chromosomal aberrations in Chinese hamster lung cells is examined. Responses of an assay are categorized as clastogenic (>10% aberrant cells) and nonclastogenic (<5% aberrant cells). Each of the compounds is represented by calculated structural descriptors that encode topological, geometric, electronic, and polar surface features. A genetic algorithm (GA) employing a k-nearest neighbor (kNN) fitness evaluator is used to iteratively search a reduced descriptor space to find small, information-rich subsets of descriptors that maximize the classification rates for clastogenic and nonclastogenic responses. To further improve modeling, a similarity measure using atom-pair descriptors is employed to create more homogeneous data subsets. Three different data sets are examined. Results for a set of 297 compounds using the GA-kNN method were 86.5% and 80.0% correct classification in the training set and prediction set, respectively. Results for a subset of 279 compounds in model 2 are 85.7% and 85.7% for the training and prediction sets, respectively. Results for a subset of 182 compounds in model 3 are 91.5% and 94.4% for the training and prediction sets, respectively. Creating smaller, more topologically similar data sets result in improved classification rates.

Physiology and endocrinology of zinc accumulation during the female squirrelfish reproductive cycle.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of hepatic zinc than any other studied animal. This accumulation is accompanied by high expression of the zinc-binding protein, metallothionein (MT), and is strongly correlated to the onset of sexual maturity. In an attempt to further characterize the timeframe of this accumulation, and to possibly discern any potential mediators, we examined the physiology and endocrinology of the yearly reproductive cycle of mature female squirrelfish. There are two separate reproductive events during the year in December-January and again in March-April, as evidenced by peaks in ovarian growth, VTG production, steroid levels, zinc accumulation and redistribution. Increased hepatic zinc seems to be preceded by a necessary increase in MT, but this was not clearly correlated to plasma 17beta-estradiol, testosterone, or progesterone levels. The plasma zinc protein vitellogenin (VTG) is one, but probably not the predominant, vehicle for the transport of hepatic zinc to the ovary.

Development of binary classification of structural chromosome aberrations for a diverse set of organic compounds from molecular structure.

Classification models are generated to predict in vitro cytogenetic results for a diverse set of 383 organic compounds. Both k-nearest neighbor and support vector machine models are developed. They are based on calculated molecular structure descriptors. Endpoints used are the labels clastogenic or nonclastogenic according to an in vitro chromosomal aberration assay with Chinese hamster lung cells. Compounds that were tested with both a 24 and 48 h exposure are included. Each compound is represented by calculated molecular structure descriptors encoding the topological, electronic, geometrical, or polar surface area aspects of the structure. Subsets of informative descriptors are identified with genetic algorithm feature selection coupled to the appropriate classification algorithm. The overall classification success rate for a k-nearest neighbor classifier built with just six topological descriptors is 81.2% for the training set and 86.5% for an external prediction set. The overall classification success rate for a three-descriptor support vector machine model is 99.7% for the training set, 92.1% for the cross-validation set, and 83.8% for an external prediction set.

Effects of 17 beta-estradiol on levels and distribution of metallothionein and zinc in squirrelfish.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.

Prediction of eye irritation from organic chemicals using membrane-interaction QSAR analysis.

Eye irritation potency of a compound or mixture has traditionally been evaluated using the Draize rabbit-eye test (Draize et al., 1944). In order to aid predictions of eye irritation and to explore possible corresponding mechanisms of eye irritation, a methodology termed "membrane-interaction QSAR analysis" (MI-QSAR) has been developed (Kulkarni and Hopfinger 1999). A set of Draize eye-irritation data established by the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) (Bagley et al., 1992) was used as a structurally diverse training set in an MI-QSAR analysis. Significant QSAR models were constructed based primarily upon aqueous solvation-free energy of the solute and the strength of solute binding to a model phospholipid (DMPC) monolayer. The results demonstrate that inclusion of parameters to model membrane interactions of potentially irritating chemicals provides significantly better predictions of eye irritation for structurally diverse compounds than does modeling based solely on physiochemical properties of chemicals. The specific MI-QSAR models reported here are, in fact, close to the upper limit in both significance and robustness that can be expected for the variability inherent to the eye-irritation scores of the ECETOC training set. The MI-QSAR models can be used with high reliability to classify compounds of low- and high-predicted eye irritation scores. Thus, the models offer the opportunity to reduce animal testing for compounds predicted to fall into these two extreme eye-irritation score sets. The MI-QSAR paradigm may also be applicable to other toxicological endpoints, such as skin irritation, where interactions with cellular membranes are likely.

Quantitative component analysis of mixtures for risk assessment: application to eye irritation.

A methodology called quantitative component analysis of mixtures (QCAM) was used to analyze an existing set of product formulation data to determine if the irritating ingredients in the mixtures could be identified. Eye irritation scores, based on a rat model, for 18 mixtures having a composite total of 37 components, were analyzed by QCAM. QCAM relates a net toxicity measure of a mixture to the toxicities of the individual components of the mixture through linear, quadratic, and pairwise cross-component concentration-dependent interactions. A correlation model is established using a particular genetic algorithm employing either multidimensional linear regression or partial least-squares regression fitting. Cornea eye irritation and average eye irritation are well-explained in terms of a linear model of, at most, three components over the set of mixtures. Moreover, extensive cornea and average eye irritations are due to only one of these three components of the mixtures. Also, one of the three significant components was predicted to decrease the extent of eye irritation, and subsequently identified as an "anti-irritant" in contact lens solutions. A reasonable linear correlation model could also be developed for conjunctiva irritation, but no significant iris irritation model could be constructed. The addition of quadratic and/or cross-component concentration terms to a linear correlation model did not statistically improve the overall resultant model. The QCAM models permit estimation of the intrinsic (self) toxicity of each of the components of a mixture, and may aid in the reduction, and ultimate elimination, of the need for animal eye irritation studies.

Genotoxicity and subchronic toxicity studies with heated olestra.

Olestra is a class of sucrose-fatty acid polyesters intended for use as a non-caloric replacement of edible oil. Genotoxicity and subchronic toxicity studies were conducted to determine whether olestra could form genotoxic or toxic breakdown products during simulated commercial use. Heated olestra was prepared for these studies by batch-frying potato slices in olestra at 177-185 degrees C for 25-32 hr over 5-7 days. Genotoxicity of this previously heated olestra was assessed in four standard in vitro assays: (1) Salmonella mutagenesis (Ames test); (2) forward mutagenesis of mouse lymphoma cells at the thymidine kinase locus; (3) unscheduled DNA synthesis in rat hepatocytes; and (4) clastogenicity in cultured Chinese hamster ovary cells. These tests were conducted with previously heated olestra at concentrations up to at least 5 mg/ml both in the absence of exogenous bioactivation and, for assays (1), (2) and (4) with added liver microsomal (S-9) activation. The Ames and mouse lymphoma assays were performed with olestra (10 mg/ml and 23 mg/litre, respectively) either alone or emulsified with the non-toxic, non-ionic surfactant Pluronics F68, both in the presence and absence of metabolic activation. To test for clastogenicity in vivo, rats were administered previously heated olestra by gavage at 5 g/kg per day for up to 5 days and bone marrow cells were examined for chromosomal aberrations. Heated olestra lacked genotoxic activity detectable by the aforementioned assays. Heated olestra was fed to Fischer 344 rats at up to 10% of the diet (w/w) for 91 days. Evaluation of survival, food consumption, feed efficiency, physical condition, body weight, organ weight, haematological and clinical chemistry parameters, and histomorphology revealed no adverse effects attributable to ingestion of heated olestra at exposure levels in excess of those anticipated for human consumption. It is concluded that olestra used as a deep-frying medium conveys no genotoxic or toxic hazard at anticipated levels of human consumption.

The low pH Syrian hamster embryo (SHE) cell transformation assay: a revitalized role in carcinogen prediction.

A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236, 933-941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964-971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160-179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291-295 and Ashby (1994) 308, 113-114; Madle (1993) Mutation Res., 300, 73-76 and Madle (1994) 308, 111-112; Zeiger (1994) Mutation Res., 304, 309-314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) cell transformation assay alone and in combination with the Salmonella mutation assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE cell transformation assay and an extensive review of the databases which support our conclusion that the SHE cell transformation assay provides an improved prediction of rodent bioassay results relative to other in vitro genotoxicity test batteries.

Assessment of the in vivo genotoxicity of 2-hydroxy 4-methoxybenzophenone.

The genotoxic potential of 2-hydroxy 4-methoxy-benzophenone (benzophenone-3, Bz-3), a commonly used sunscreen, has been evaluated previously with in vitro systems. Data from Salmonella studies (with and without activation) have been predominantly negative, but two reports have shown weakly positive results in a single bacterial strain under conditions of metabolic activation. In addition, Bz-3 has been reported to induce chromosome aberrations and equivocal results for sister chromatid exchange in Chinese hamster ovary (CHO) cells. We used the Drosophila somatic mutation and recombination test (SMART) and in vivo cytogenetics in rat bone marrow to define the potential for in vivo expression of this in vitro activity. For the SMART assay, larva from a mating of "multiple wing hair" (mwh) females with heterozygous "flare" (flr) males were exposed to 0, 3000, or 3500 ppm Bz-3 or 25 ppm dimethylnitrosamine (DMN, positive control) for 72 hr. A recombination between the mwh and flr genes produces twin wing spots, while events such as deletions produce single spots. None of the Bz-3-treated larva produced flies with significantly more single or multiple wing spots than controls. In contrast, DMN-treated larva produced flies with significantly more single or multiple wing spots than controls. The in vivo cytogenetic assay in rat bone marrow cells was conducted to evaluate the clastogenicity of Bz-3. Sprague-Dawley rats were treated by oral gavage with a single administration of 0.0, 0.5, 1.67, or 5 gm/kg Bz-3 or a single dose of 5 gm/kg/day Bz-3 for 5 consecutive days. Cyclophosphamide (CP) was the positive control and was administered at 20 mg/kg with both treatment regimens. Colchicine growth-arrested bone marrow cells were collected 8 and 12 hr after the single treatment and 12hr after the last daily treatment. Under either treatment protocol none of the Bz-3 concentrations caused any significant increase in chromosomal aberrations. Results from these two studies strongly support the conclusion that Bz-3 is not genotoxic in vivo.

Overview of the workshops on statistical analysis of mutation data from transgenic mice.

Early results from two transgenic mouse mutation assays, Big Blue [Kohler SW et al. (1991): Proc Natl Acad Sci USA 88:7958-7962] and Muta Mouse [Myhr BC (1991): Environ Mol Mutagen 18:308-315], raised questions about appropriate study design and methods for statistical analysis. First, there were a number of potential sources of variability in the technical aspects of the assay. These are "how we do it in our laboratory" differences, which, if not controlled, ultimately make inter-laboratory comparison of data difficult. Second, separate from the technical sources of variability were a number of study design issues, e.g., how many animals are needed in each treatment group, how many times should the animal be dosed, what is the appropriate expression period for a particular tissue, how many plaques need to be collected from each animal, and so on. To address these questions and to identify and understand the sources of variability in mutation data from these systems, a workshop was held in June, 1992 in Cincinnati, Ohio, USA. A core group of biologists and statisticians discussed possible sources of bias (tissue sampling, phage recovery and transgene recovery), possible sources of variability in mutant frequency (between animals, protocol-based sources, and design-based sources) and assay sensitivity. Following two days of discussion on protocol design and assay procedures, three action steps were recommended: (1) compile a data base of existing mutation data in transgenic mice to study its statistical features, (2) develop standard protocols for the mutation assays; and (3) use the standard protocol to generate a large data base of mutant frequencies in liver DNA from untreated mice for statistical study and analysis. This report summarizes the proceedings and recommendations of the workshop. The progress made toward these recommendations was reviewed in a second workshop, held in April, 1993, in Norfolk, Virginia, part of which is the subject of the accompanying paper by Piegorsch et al. To date, a standard protocol has been developed for the Big Blue mutagenesis assay and a data base of over 90 million plaques from seven labs using either the Big Blue or Muta Mouse system has been assembled, including a large data set of spontaneous liver mutant frequencies in the Big Blue system.

Sources of variability in data from a lacI transgenic mouse mutation assay.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.

Lack of genotoxicity of cross-linked acrylate polymers in four short-term genotoxicity assays.

Three cross-linked polyacrylate polymers containing either methylenebis-acrylamide (MBA), trimethylolpropane triacrylate (TMPTA), or triallylamine (TAA) cross-linkers were tested for genotoxicity with the Salmonella mammalian microsome assay, the L5178Y mouse lymphoma TK +/- assay, the unscheduled DNA synthesis assay in primary cultures of rat hepatocytes, and the in vivo bone marrow cytogenetic assay. The results indicate that none of the three polymers was genotoxic in these assays.

Characterization of a method for quantitating food consumption for mutation assays in Drosophila.

Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period (Thompson and Reeder: Environmental Mutagenesis 10:357-365, 1987). Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.

Evaluation of olestra in short-term genotoxicity assays.

Olestra, a mixture of hexa-, hepta- and octa-esters formed from the reaction of sucrose with long-chain fatty acids, was evaluated for its genotoxic potential in the Salmonella/mammalian microsome test, the L5178Y thymidine kinase (TK+/-) mouse lymphoma assay, an unscheduled DNA synthesis assay in primary rat hepatocytes, and an in vitro cytogenetic assay in Chinese hamster ovary cells. The results indicated that olestra was non-genotoxic in these assays.

Genotoxicity of zinc in 4 short-term mutagenicity assays.

The genotoxicity of zinc was examined in 4 short-term mutagenicity assays. Zinc acetate produced dose-related positive responses in the L5178Y mouse lymphoma assay and an in vitro cytogenetic assay with Chinese hamster ovary cells, but was negative in the Salmonella mutation assay and did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. Zinc-2,4-pentanedione produced frameshift mutations in Salmonella tester strains TA1538 and TA98, but did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. The effect of ligand binding of zinc in the in vitro test systems is discussed.

Lack of genotoxicity with acrylate polymers in five short-term mutagenicity assays.

Two linear polymers of acrylic acid (average molecular weight = 2,000 and 4,500) and one linear copolymer of acrylic and maleic acids (average molecular weight = 12,000) were tested for mutagenicity with the Salmonella mammalian microsome assay, the L5178Y mouse lymphoma assay at the TK locus, an unscheduled DNA synthesis assay in primary cultures of rat hepatocytes, and either an in vitro cytogenetic assay with CHO cells or the bone marrow micronucleus assay in mice. The results of all the assays were uniformly negative for the three polymers.

Design and simulated performance of a CARS spectrometer for dynamic temperature measurements using electronic heterodyning.

A new design for generating CARS signals and for the detection and processing of these signals is presented and evaluated. The design is based on electronic heterodyning of the CARS spectrum of nitrogen at two selected narrowband frequencies, ratioing the resulting signal strengths, and comparing this ratio with a theoretically derived temperature scale. A reference cell is incorporated into the design for system calibration and for accurate temperature measurements. The spectrometer is found capable of measuring temperature in the submillisecond time scale with an accuracy of 10% in the 1000-2000 K temperature range. A typical result using the Hg(x)Cd(1-x) Te photomixer for T = 1500 K,DeltaT = 50 K is a SNR of 21 dB and a data collection rate of 300 Hz.