William W. West

A Prospective, Multi-institutional, Pathologist-Based Assessment of 4 Immunohistochemistry Assays for PD-L1 Expression in Non–Small Cell Lung Cancer

Importance Four assays registered with the US Food and Drug Administration (FDA) detect programmed cell death ligand 1 (PD-L1) to enrich for patient response to anti–programmed cell death 1 and anti–PD-L1 therapies. The tests use 4 separate PD-L1 antibodies on 2 separate staining platforms and have their own scoring systems, which raises questions about their similarity and the potential interchangeability of the tests. Objective To compare the performance of 4 PD-L1 platforms, including 2 FDA-cleared assays, 1 test for investigational use only, and 1 laboratory-developed test. Design, Setting, and Participants Four serial histologic sections from 90 archival non–small cell lung cancers from January 1, 2008, to December 31, 2010, were distributed to 3 sites that performed the following immunohistochemical assays: 28-8 antibody on the Dako Link 48 platform, 22c3 antibody on the Dako Link 48 platform, SP142 antibody on the Ventana Benchmark platform, and E1L3N antibody on the Leica Bond platform. The slides were scanned and scored by 13 pathologists who estimated the percentage of malignant and immune cells expressing PD-L1. Statistical analyses were performed from December 1, 2015, to August 30, 2016, to compare antibodies and pathologists’ scoring of tumor and immune cells. Main Outcomes and Measures Percentages of malignant and immune cells expressing PD-L1. Results Among the 90 samples, the SP142 assay was an outlier, with a significantly lower mean score of PD-L1 expression in both tumor and immune cells (tumor cells: 22c3, 2.96; 28-8, 3.26; SP142, 1.99; E1L3N, 3.20; overall mean, 2.85; and immune cells: 22c3, 2.15; 28-8, 2.28; SP142, 1.62; E1L3N, 2.28; overall mean, 2.08). Pairwise comparisons showed that the scores from the 28-8 and E1L3N tests were not significantly different but that the 22c3 test showed a slight (mean difference, 0.24-0.30) but statistically significant reduction in labeling of PD-L1 expression in tumor cells. Evaluation of intraclass correlation coefficients (ICCs) between antibodies to quantify interassay variability for PD-L1 expression in tumor cells showed high concordance between antibodies for tumor cell scoring (0.813; 95% CI, 0.815-0.839) and lower levels of concordance for immune cell scoring (0.277; 95% CI, 0.222-0.334). When examining variability between pathologists for any single assay, the concordance between pathologists’ scoring for PD-L1 expression in tumor cells ranged from ICCs of 0.832 (95% CI, 0.820-0.844) to 0.882 (95% CI, 0.873-0.891) for each assay, while the ICCs from immune cells for each assay ranged from 0.172 (95% CI, 0.156-0.189) to 0.229 (95% CI, 0.211-0.248). Conclusions and Relevance The assay using the SP142 antibody is an outlier that detected significantly less PD-L1 expression in tumor cells and immune cells. The assay for antibody 22c3 showed slight yet statistically significantly lower staining than either 28-8 or E1L3N, but this significance was detected only when using the mean of 13 pathologists’ scores. The pathologists showed excellent concordance when scoring tumor cells stained with any antibody but poor concordance for scoring immune cells stained with any antibody. Thus, for tumor cell assessment of PD-L1, 3 of the 4 tests are concordant and reproducible as read by pathologists.

Serum enzyme levels during intestinal ischemia

Because the intestinal mucosa is most sensitive to ischemia, serum levels of mucosal enzymes, such as diamine oxidase, may be most likely to indicate intestinal ischemia. Our aim was to compare serum levels of mucosal (diamine oxidase, alkaline phosphatase) and seromuscular (creatinine phosphokinase, lactic dehydrogenase, serum glutamic oxaloacetic transminase) enzymes during intestinal ischemia of varying extent and duration in dogs. Group 1 (n = 6) underwent sham laparotomy. Group 2 (n = 8) had 50% of the small intestine devascularized. Group 3 (n = 8) had the superior mesenteric artery occluded for 2 hours and released. Group 4 (n = 8) had the superior mesenteric artery ligated. Serum samples were obtained before and 2, 4, 8, and 24 hours after operation, and histologic specimens were examined at 4 hours. Creatinine phosphokinase levels became elevated within 4 hours of ischemic injury in group 2 (223 +/- 197 vs. 68 +/- 26, p less than 0.05) and group 4 (212 +/- 136 vs. 76 +/- 29, p less than 0.05). Significant elevation of serum enzymes levels, except diamine oxidase, occurred in groups 2, 3, and 4 at 24 hours, including those with normal histology after temporary superior mesenteric artery occlusion. Thus seromuscular enzymes, particularly creatinine phosphokinase, were more likely to be elevated during intestinal ischemia. Enzyme levels were not influenced by the extent and reversibility of the ischemic injury.

Intranasal organic dust exposure-induced airway adaptation response marked by persistent lung inflammation and pathology in mice

Organic dust exposure in agricultural environments results in an inflammatory response that attenuates over time, but repetitive exposures can result in chronic respiratory disease. Animal models to study these mechanisms are limited. This study investigated the effects of single vs. repetitive dust-induced airway inflammation in mice by intranasal exposure method. Mice were exposed to swine facility dust extract (DE) or saline once and once daily for 1 and 2 wk. Dust exposure resulted in increased bronchoalveolar lavage fluid neutrophils and macrophages after single and repetitive exposures. Lavage fluid TNFalpha, IL-6, keratinocyte chemoattractant, and macrophage inflammatory protein-2 were significantly increased after single and repetitive dust exposures, but were dampened in 2-wk dust-exposed mice compared with single exposure. Dust exposure induced PKCalpha and -epsilon activation in isolated tracheal epithelial cells but were dampened with repetitive exposures. Ex vivo stimulation of alveolar macrophages from 2-wk animals demonstrated reduced cytokine responsiveness and phagocytic ability. Significant lung pathology occurred with development of mixed mononuclear cellular aggregates (T and B lymphocytes, phagocytes) after repetitive dust exposure, a novel observation. Airway hyperresponsiveness to methacholine occurred after single dust exposure but resolved after 2 wk. Collectively, intranasal exposure to DE results in significant lung inflammatory and pathological responses marked by a modulated innate immune response to single and repetitive dust exposures that is associated with PKC activity.

DECREASED EXPRESSION OF CELLULAR PROSTATIC ACID PHOSPHATASE INCREASES TUMORIGENICITY OF HUMAN PROSTATE CANCER CELLS

PURPOSE Understanding cell proliferation regulation in hormone refractory prostate cancer may provide answers for novel solutions. Protein tyrosine phosphatases have been thought to have key roles in regulating cell proliferation and be involved in oncogenesis, although to our knowledge their functional roles in human prostate cancer remain unknown. Human prostatic acid phosphatase (PAcP), a major phosphatase in prostate epithelium, has been shown to function as a neutral protein tyrosine phosphatase in these cells. We evaluated the biological significance of cellular prostatic acid phosphatase expression in human prostate cancer cells. MATERIALS AND METHODS Immunohistochemical testing of human prostate cancer archival specimens was done to evaluate the expression of cellular PAcP. Immunoprecipitation and immunoblotting were performed to determine cellular PAcP and SH2 domain-bearing tyrosine phosphatase-1 levels as well as tyrosine phosphorylation of c-ErbB-2/neu in different human prostate cancer cells. The biological behavior of LNCaP derivative sublines was characterized in vitro and in vivo by soft agar analysis and xenograft animal inoculation. RESULTS Immunohistochemical staining of human prostate clearly showed that cellular levels of PAcP significantly decreases in prostate cancer cells (p <0.001). The results of biochemical characterization revealed that the cellular level of PAcP but not SHP-1, another differentiation associated protein tyrosine phosphatase, consistently correlated negatively with the growth of several human prostate cancer cell lines. Reintroducing cellular PAcP activity in prostate cancer cells by PAcP complementary DNA transfection resulted in decreased tyrosine phosphorylation of c-ErbB-2/neu, decreased proliferation rates in culture as well as decreased anchorage independent growth in soft agar. The xenograft animal model demonstrated that a higher tumor growth rate as well as larger size is associated with a lower level of cellular PAcP. CONCLUSIONS Cellular PAcP can down-regulate prostate cancer cell growth, at least partially by dephosphorylating c-ErbB-2/neu. Therefore, decreased cellular PAcP expression in cancer cells may be involved in prostate cancer progression.

Toll-Like Receptor 2 Regulates Organic Dust–Induced Airway Inflammation

Organic dust exposure in agricultural environments results in significant airway inflammatory diseases. Gram-positive cell wall components are present in high concentrations in animal farming dusts, but their role in mediating dust-induced airway inflammation is not clear. This study investigated the role of Toll-like receptor (TLR) 2, a pattern recognition receptor for gram-positive cell wall products, in regulating swine facility organic dust extract (DE)-induced airway inflammation in mice. Isolated lung macrophages from TLR2 knockout mice demonstrated reduced TNF-α, IL-6, keratinocyte chemoattractant/CXCL1, but not macrophage inflammatory protein-2/CXCL2 expression, after DE stimulation ex vivo. Next, using an established mouse model of intranasal inhalation challenge, we analyzed bronchoalveolar lavage fluid and lung tissue in TLR2-deficient and wild-type (WT) mice after single and repetitive DE challenge. Neutrophil influx and select cytokines/chemokines were significantly lower in TLR2-deficient mice at 5 and 24 hours after single DE challenge. After daily exposure to DE for 2 weeks, there were significant reductions in total cellularity, neutrophil influx, and TNF-α, IL-6, CXCL1, but not CXCL2 expression, in TLR2-deficient mice as compared with WT animals. Lung pathology revealed that bronchiolar inflammation, but not alveolar inflammation, was reduced in TLR2-deficient mice after repetitive exposure. Airway hyperresponsiveness to methacholine after dust exposure was similar in both groups. Finally, airway inflammatory responses in WT mice after challenge with a TLR2 agonist, peptidoglycan, resembled DE-induced responses. Collectively, these results demonstrate that the TLR2 pathway is important in regulating swine facility organic dust-induced airway inflammation, which suggests the importance of TLR2 agonists in mediating large animal farming-induced airway inflammatory responses.

Concordance between whole-slide imaging and light microscopy for routine surgical pathology.

The use of high-resolution digital images of histopathology slides as a routine diagnostic tool for surgical pathology was investigated. The study purpose was to determine the diagnostic concordance between pathologic interpretations using whole-slide imaging and standard light microscopy. Two hundred fifty-one consecutive surgical pathology cases (312 parts, 1085 slides) from a single pathology service were included in the study after cases had been signed out and reports generated. A broad array of diagnostic challenges and tissue sources were represented, including 52 neoplastic cases. All cases were digitized at ×20 and presented to 2 pathologists for diagnosis using whole-slide imaging as the sole diagnostic tool. Diagnoses rendered by the whole-slide imaging pathologists were compared with the original light microscopy diagnoses. Overall concordance between whole-slide imaging and light microscopy as determined by a third pathologist and jury panel was 96.5% (95% confidence interval, 94.8%-98.3%). Concordance between whole-slide imaging pathologists was 97.7% (95% confidence interval, 94.7%-99.2%). Five cases were discordant between the whole-slide imaging diagnosis and the original light microscopy diagnosis, of which 2 were clinically significant. Discordance resulted from interpretive criteria or diagnostic error. The whole-slide imaging modality did not contribute to diagnostic differences. Problems encountered by the whole-slide imaging pathologists primarily involved the inability to clearly visualize nuclear detail or microscopic organisms. Technical difficulties associated with image scanning required at least 1 slide be rescanned in 13% of the cases. Technical and operational issues associated with whole-slide imaging scanning devices used in this study were found to be the most significant obstacle to the use of whole-slide imaging in general surgical pathology.

Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types—a K5+/K19− type and a K5+/K19+ type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

Intestinal diamine oxidase levels reflect ischemic injury

Mucosal diamine oxidase (DAO) decreases during intestinal ischemia and may be a useful marker of intestinal ischemic injury. Tissue DAO activity and histologic changes were studied in intestinal segments taken from the midpoint of the small intestine before and 2, 4, and 24 hr after manipulation of the intestinal blood supply in 24 mongrel dogs. Intestinal DAO activity decreased significantly (17 +/- 21% of control value) 24 hr after SMA ligation and was associated with abnormal histology (histology score 7.8 +/- 2.9 at 24 hr vs 0.3 +/- 0.5 at 0 hr). SMA occlusion for 2 hr resulted in a significant decrease in DAO activity (45 +/- 36% of control value) 4 hr after manipulation which returned to normal at 24 hr, as did the histologic injury. Ligation of both the mesenteric arteries and veins resulted in a more rapid decrease in DAO activity. Decreased DAO activity correlated with the extent of histologic injury. Intestinal ischemia is associated with decreased intestinal DAO activity, which is influenced by the mechanism and duration of intestinal ischemia.

DIFFERENTIAL IN VIVO EFFECTS OF WHOLE CIGARETTE SMOKE EXPOSURE VERSUS CIGARETTE SMOKE EXTRACT ON MOUSE CILIATED TRACHEAL EPITHELIUM

In this study the authors compared the affect of vapor phase cigarette smoke (CS) versus cigarette smoke extract (CSE) on the lungs and upper airway of C57BL/6 mice. The authors found that CSE treatment significantly increased neutrophil influx (P < .001), baseline ciliary beat frequency (CBF) (P < .05), and protein kinase C activity compared to CS and controls. Isoproterenol increased CBF with CS exposure, but decreased CBF with CSE (P < .01). Isoproterenol increased protein kinase A (PKA) activity in all groups except CSE. CSE exposure induced inflammatory cell bronchiolitis. These data indicate that CSE exposure has differential effects on the lungs and tracheal epithelium compared to CS exposure.

αβ T Cells and a Mixed Th1/Th17 Response are Important in Organic Dust-Induced Airway Disease

BACKGROUND Organic dust exposure in agricultural environments induces an inflammatory response that attenuates over time, yet repetitive dust exposures result in chronic lung diseases. Animal models resembling this chronic lung inflammatory response have been developed, yet the underlying cellular mechanisms are not well defined. OBJECTIVE Because mice repetitively exposed to organic dust extracts (DE) display increased CD3+ T cell lung infiltrates, we sought to determine the phenotype and importance of these cells. METHODS Mice received swine confinement DE repetitively for 3 weeks by established intranasal inhalation protocol. Studies were conducted with peptidoglycan (PGN) because it is a major DE component in large animal farming environments and has shared similar biologic effects with DE. Enumeration of T cells and intracellular cytokine profiles were conducted by flow cytometry techniques. Whole lung homogenate cytokines were analyzed by multiplex immunoassay. T cell receptor (TCR) αβ knockouts were used to determine the functional importance of αβ-expressing T cells. RESULTS DE increased lung-associated CD3+CD4+ T cells and interleukin (IL)-17 (but not IL-4, interferon [IFN]-γ, IL-10) producing CD4+ T cells. PGN treatment resulted in increased IL-17 and IFN-γ producing CD4+ T cells and IFN-γ producing CD8+ T cells. Both DE and PGN augmented expression of cytokines associated with Th1 and Th17 polarization in lung homogenates. DE-induced lung mononuclear aggregates and bronchiolar compartment inflammation were significantly reduced in TCR knockout animals; however, neutrophil influx and alveolar compartment inflammation were not affected. CONCLUSION Studies demonstrated that DE and PGN exposure promote a Th1/Th17 lung microenvironment and that αβ-expressing T cells are important in mediating DE-induced lung pathologic conditions.