Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens.

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell-cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.

Characterizing Intra-Tumor and Inter-Tumor Variability of Immune Cell Infiltrates in Murine Syngeneic Tumors.

Murine tumors are indispensable model systems in preclinical immuno-oncology research. While immunologic heterogeneity is well-known to be an important factor that can influence treatment outcome, there is a severe paucity of data concerning the nature of this heterogeneity in murine tumor models. Using serial sectioning methodology combined with IHC analysis and whole-slide image analysis, the depth-dependent variation in immune-cell abundance in tumor specimens was investigated at single-cell resolution. Specifically, intra- and intertumor variability in cell density of nine immune-cell biomarkers was quantified in multiple murine tumor models. The analysis showed that intertumor variability was typically the dominant source of variation in measurements of immune-cell densities. Statistical power analysis revealed the effect of group size and variance in immune-cell density on the predictive power of detecting a statistically meaningful fold-change in immune-cell density. Intertumor variability in the ratio of immune-cell densities showed distinct patterns in select tumor models and revealed the existence of strong correlations between select biomarker pairs. Furthermore, the relative proportion of immune cells at different depths across tumor samples was preserved in some but not all tumor models, thereby revealing the existence of compositional heterogeneity. Taken together, these results reveal novel insights into the nature of immunologic heterogeneity, which is not accessible through typical omics approaches.

Alcohol exposure in utero increases susceptibility to prostate tumorigenesis in rat offspring.

BACKGROUND: Prenatal alcohol exposure has been shown to increase offspring susceptibility to some chemical carcinogens. Whether prenatal exposure to alcohol makes the offspring more susceptible to the development of prostate cancer is not known. Therefore, we determined whether any functional abnormalities and increased cancer susceptibility exist in the prostate of fetal alcohol-exposed male rats during the adult period. METHODS: Pregnant rats were fed with a liquid diet containing alcohol (alcohol-fed [AF]), or pair-fed with isocaloric liquid diet (PF) or ad libitum fed with rat chow (ad lib-fed). Male offspring of these rats were given N-Nitroso-N-methylurea and testosterone to induce prostate neoplasia or left untreated. Around 6 to 8 months of age, the prostates of these animals were processed for determination of biochemical changes and histopathologies. RESULTS: Prostates of noncarcinogen treated animals that were alcohol exposed during the prenatal period demonstrated inflammatory cell infiltration and epithelial atypia and increased number of proliferative cells in the ventral lobe of this gland, but the prostate of control animal showed normal cytoarchitecture. In addition, prenatal alcohol-exposed rats showed decreased levels of cell-cell adhesion marker and increased estrogenic activity in the ventral prostate. Prenatally ethanol (EtOH)-exposed rats, when treated with carcinogen and testosterone, showed histological evidence for high-grade prostatic intraepithelial neoplasia (PIN) primarily in the ventral prostate, whereas control animals showed only low-grade PIN. Prenatally EtOH-exposed rats treated with carcinogen and testosterone also showed increased number of proliferative cells and androgen receptor with concomitant decreased levels of tumor suppressor proteins in the ventral prostate. CONCLUSIONS: These results suggest for the first time that prenatal EtOH exposures induce histophysiological changes in the prostate as well as it increases the susceptibility of the prostate to develop neoplasia during adulthood.

Clinicopathologic relevance of apoptotic and proliferative factors in human lung adenocarcinoma: Fas expression correlates with the histologic subtype, but not with the degree of apoptosis.

We immunohistochemically examined 141 surgically resected peripheral lung adenocarcinomas for the expression of Fas, single stranded (ss-) DNA and Ki-67, and statistically evaluated the relationship of these parameters with other clinicopathologic variables, including clinical stage, nodal involvement, and histopathologic subtypes classified according to WHO criteria. Fas expression by cancer cells was characteristically localized in the cytoplasm, and the extent of expression correlated well with the degree of Ki-67 reactivity (p = 0.0004), but not with the degree of apoptic occurrence, as assessed by ss-DNA reactivity. Cancer cells of the bronchioloalveolar carcinoma (BAC) subtype without invasive growth exhibited a significantly lower Fas expression than those of other subtypes (p < 0.0001). Positive expression of Fas was frequently associated with a high incidence of nodal involvement and advanced clinical stage, as compared with cases of negative expression (p = 0.0111 and p = 0.0439, respectively). Multivariate analysis revealed that Fas expression significantly correlated with the histologic subtype, but not with tumor size, nodal involvement, or clinical stage. Survival analysis determined by the log-rank test revealed that clinical stage and Ki-67 reactivity were poor prognostic variables, and Fas expression was not statistically significant. Based on these data, intracytoplasmic expression of Fas in cancer cells may participate in the development of resistance to fas-mediated apoptosis.

Lung adenocarcinoma with bronchioloalveolar carcinoma component is frequently associated with foci of high-grade atypical adenomatous hyperplasia.

We assessed the occurrence of atypical adenomatous hyperplasia (AAH) in whole lung lobes with primary cancer lesions. Following surgical resection, tissue specimens were sliced to a thickness of 4 mm (3,641 specimens from 61 cases; mean = 59.7 specimens per case). A total of 119 AAH foci were found and an association was evident in 25 (57%) of 44 adenocarcinomas, 3 (30%) of 10 squamous cell carcinomas, and 2 (29%) of 7 other lung cancers. Histologic evaluation showed that 108 AAH foci were categorized as low-grade and the other 11 as high-grade AAH. These 11 foci of high-grade AAH were present in 7 patients with adenocarcinoma, and in 1 patient there was a synchronous double primary lung adenocarcinoma. High-grade AAH was closely associated with bronchioloalveolar carcinoma (BAC) type adenocarcinoma, and low-grade AAH with non-BAC adenocarcinoma. The mean +/- SD Ki-67 labeling index in high-grade AAH (3.5%+/-2.9%) was significantly higher than for the low-grade index (1.4%+/-1.6%). We propose that foci of high- but not low-grade AAH may be potential precursor lesions of lung adenocarcinoma, especially with the BAC component.