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.

Mutagenicity testing of diethylene glycol monobutyl ether.

The mutagenic potential of diethylene glycol monobutyl ether (diEGBE) was examined with a Tier I battery of in vitro assays followed by a Tier II in vivo Drosophila sex-linked recessive lethal assay. The in vitro battery consisted of: the Salmonella mutagenicity test, the L5178Y mouse lymphoma test, a cytogenetics assay using Chinese hamster ovary cells and the unscheduled DNA synthesis (UDS) assay in rat hepatocytes. Results of the Salmonella mutagenicity test, the cytogenetics test, and the rat hepatocyte assay were negative at concentrations up to 20 microL/plate, 7.92 microL/mL, and 4.4 microL/mL, respectively. Toxicity was clearly demonstrated at all high doses. A weak, but dose-related increase in the mutation frequency (4-fold increase over the solvent control at 5.6 microL/mL with 12% survival) was obtained in the L5178Y lymphoma test in the absence of metabolic activation. Results of the mouse lymphoma assay were negative in the presence of the S-9 activation system. The significance of the mouse lymphoma assay were negative in the presence of the S-9 activation system. The significance of the mouse lymphoma assay results were assessed by performing the Tier II sex-linked recessive lethal assay in Drosophila in which the target tissue is maturing germinal cells. Both feeding (11,000 ppm for 3 days) and injection (0.3 microL of approximately 14,000 ppm solution) routes of administration were employed in the Drosophila assay. Approximately 11,000 individual crosses with an equal number of negative controls were performed for each route of administration. diEGBE produced no increase in recessive lethals under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thrombin on the fibronectin-containing extracellular matrix.

The effects of human alpha-thrombin on the incorporation of exogenous human fibronectin into the extracellular matrix of cultured human skin fibroblasts were studied. Thrombin had no effect on the saturable cell-surface receptor for soluble fibronectin. Thrombin, however, did digest fibronectin bound reversibly to the cell surface and fibronectin incorporated irreversibly into the extracellular matrix. The products of thrombin digestion of cell surface and matrix-bound fibronectin were similar to the products of thrombin digestion of soluble fibronectin. These results indicate that fibronectin can potentially modulate some of the effects of thrombin on cells.

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.

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.

Method for selecting exposure levels for the Drosophila sex-linked recessive lethal assay.

The use of the Drosophila sex-linked recessive lethal assay for detecting mutagenicity of chemicals is well established. When compounds are tested by feeding adult flies, the National Toxicology Program protocol specifies a 3-day feeding regimen at an exposure level that produces about 30% mortality. Uptake of the test compound is monitored by feeding behavior, amount of excretion, or abdomen size (Woodruff et al: Environ Mutat 7:677-702, 1985). An alternate method for determining uptake is to add radiolabeled sucrose to the feeding solution and then to determine the amount of radioactivity in the flies (Gollapudi et al: Mutat Res 144:13-17, 1985). We have found that the addition of radiolabeled sucrose underestimates consumption for feeding exposures longer than 24 hr because sucrose is metabolized and as much as 30% of the label is excreted, presumably as 14CO2 or 3H2O. Here we describe a method for determining uptake of chemicals by adding 14C-leucine to the feeding solution. The incorporation of 14C-leucine is essentially linear over the 3-day feeding period, which permits accurate estimates of food consumption. Use of this method demonstrates that lower exposure levels of a chemical that do not produce mortality actually results in higher consumption by the flies. The method is proposed as a prescreen to select the appropriate exposure level for the sex-linked recessive lethal assay.

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.

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.

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.

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.

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.

GLC determination of quinidines in human plasma.

Human plasma was made alkaline and extracted with methylene chloride. To the extract was added the internal standard, cinchonine, followed by evaporation to dryness. The resultant residue was dissolved in a methanolic solution containing trimethylanilinium hydroxide. This solution was assayed by GLC for quinidines (quinidine and hydroquinidine). Evaluation of the method over a 0.5-10-mug/ml range in human plasma gave an overall precision and accuracy of +/- 4.5% (RSD and RE). Plasma of several patients was analyzed by the present method as well as by a fluorometric method for the level of quinidines. Results from the two methods were comparable.

High-pressure liquid chromatographic--mass spectrometric determination of delta9-tetrahydrocannabinol in human plasma following marijuana smoking.

A method was developed for analyzing delta9-tetrahydrocannabinol (I), a psychotomimetic constituent found in marijuana smoke. The developed method utilizes a high-pressure liquid chromatographic (HPLC) gradient elution program to separate I from the other major cannabinoids in marijuana smoke. To achieve the sensitivity required to detect I in human plasma following marijuana smoking, a mass spectrometric quantification method was developed to analyze the HPLC eluant. To 1 ml of human plasma was added a known amount of internal standard, trideuterated I. This stable isotope provided a check on extraction efficiency, a marker for UV monitoring of the HPLC effluent and subsequent collection, and a convenient mass for mass spectrometric quantification. An ion-counting technique was used in conjunction with the peak matching accessory of the mass spectrometer to provide for a rapid comparison between molecular ions of I and the internal standard. The method was linear, accurate, and reproducible over the concentration range expected for I in plasma following marijuana smoking; 2.5 ng/ml was the lower practical limit of detection. Plasma from 11 male subjects was analyzed by the method at appropriate intervals up to 24 hr after the smoking of a marijuana cigarette containing 10.8 mg of I. Results demonstrated that levels of I could be determined accurately in the plasma of marijuana smokers in the 1-hr period following smoking.

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.

Locus specificity of mutagenicity of 2,4-diaminotoluene in both L5178Y mouse lymphoma and AT3-2 Chinese hamster ovary cells.

2,4-Diaminotoluene, a hepatocarcinogenic aromatic amine, was tested for mutagenic potential at both the autosomal tk locus and the sex-linked hgprt locus of both L5178Y 3.7.2C mouse lymphoma cells and Chinese hamster ovary (CHO) AT3-2 cells. This compound was mutagenic in both cell types at the tk locus but not at the hgprt locus. Mutagenic activity was observed in L5178Y cells only in the absence of exogenous metabolic activation, but was observed in CHO-AT3-2 cells both with and without activation.

Mutagenicity of alkyl glycidyl ethers in three short-term assays.

The mutagenic potential of glycidol and 7 alkyl glycidyl ethers having straight alkyl chains of 2, 4, 6, 8, 10, 12, and 14 carbon atoms were examined in a battery of in vitro assays. The battery consisted of the Salmonella/mammalian microsome assay, the L5178Y mouse lymphoma assay, and unscheduled DNA synthesis using W138 cells. The mutagenic potential of the compounds ranged from strongly mutagenic to non-mutagenic; glycidol exhibited the greatest activity. All the ethers through C-4 showed a definite response whole the C-8 or higher ethers showed very weak or no responses. Dose-response curves were obtained by all 3 assays for those compounds that exhibited mutagenic activity. The sensitivity of each assay is discussed, as are the effects of the liver microsome systems used for metabolic activation.

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.

A method for determining the maximum tolerated dose for acute in vivo cytogenetic studies.

A method for establishing a maximum tolerated dose for use in in vivo cytogenetic studies is proposed. Probit analyses were performed on acute (ip and po) LD50 studies for triethylenemelamine, chlorambucil, methyl methanesulphonate, glycidol, phenol, Triton X-15, and dimethylsulphoxide. The concentrations corresponding to the calculated LD30, LD10 and LD1 values were given both ip and po to groups of female and male rats. Half of the animals in each group were killed about 20 hr after treatment for bone marrow cytogenetic analyses, and body weights were recorded for 10 days for the other half. The following conclusions were drawn: (1) LD50 values and consequently the maximum tolerated dose (MTD) values differ for males and females; (2) the mitotic index is not a reliable indicator of toxicity; (3) the LD1 value approximates the MTD for these compounds; (4) this value and hence the MTD is not a fixed percentage of the LD50 value; (5) body-weight changes appear to be an accurate parameter for determining whether or not an animal received an MTD level.

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.