Heterogeneous antiretroviral drug distribution and HIV/SHIV detection in the gut of three species.

HIV replication within tissues may increase in response to a reduced exposure to antiretroviral drugs. Traditional approaches to measuring drug concentrations in tissues are unable to characterize a heterogeneous drug distribution. Here, we used mass spectrometry imaging (MSI) to visualize the distribution of six HIV antiretroviral drugs in gut tissue sections from three species (two strains of humanized mice, macaques, and humans). We measured drug concentrations in proximity to CD3+ T cells that are targeted by HIV, as well as expression of HIV or SHIV RNA and expression of the MDR1 drug efflux transporter in gut tissue from HIV-infected humanized mice, SHIV-infected macaques, and HIV-infected humans treated with combination antiretroviral drug therapy. Serial 10-µm sections of snap-frozen ileal and rectal tissue were analyzed by MSI for CD3+ T cells and MDR1 efflux transporter expression by immunofluorescence and immunohistochemistry, respectively. The tissue slices were analyzed for HIV/SHIV RNA expression by in situ hybridization and for antiretroviral drug concentrations by liquid chromatography-mass spectrometry. The gastrointestinal tissue distribution of the six drugs was heterogeneous. Fifty percent to 60% of CD3+ T cells did not colocalize with detectable drug concentrations in the gut tissue. In all three species, up to 90% of HIV/SHIV RNA was found to be expressed in gut tissue with no exposure to drug. These data suggest that there may be gut regions with little to no exposure to antiretroviral drugs, which may result in low-level HIV replication contributing to HIV persistence.

Racial Differences in PAM50 Subtypes in the Carolina Breast Cancer Study.

Background: African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood. Methods: Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests. Results: Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women. Conclusions: Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases.

Ki-67 Expression in Breast Cancer Tissue Microarrays: Assessing Tumor Heterogeneity, Concordance With Full Section, and Scoring Methods.

OBJECTIVES: Ki-67 has been proposed to be used as a surrogate marker to differentiate luminal breast carcinomas (BCs). The purpose of this study was to determine the utility of and best approaches for using tissue microarrays (TMAs) and Ki-67 staining to distinguish luminal subtypes in large epidemiology studies of luminal/human epidermal growth factor receptor 2 (HER2)-negative BC. METHODS: Full-section and TMA (three 0.6-mm cores and two 1.0-mm cores) slides of 109 cases were stained with Ki-67 antibody. We assessed two ways of collapsing TMA cores: a weighted approach and mitotically active approach. RESULTS: For cases with at least a single 0.6-mm TMA core (n = 107), 16% were misclassified using a mitotically active approach and 11% using a weighted approach. For cases with at least a single 1.0-mm TMA core (n = 101), 5% were misclassified using either approach. For the 0.6-mm core group, there were 33.3% discordant cases. The number of discordant cases increased from 18% in the group of two cores to 40% in the group of three cores (P = .039). CONCLUSIONS: Ki-67 tumor heterogeneity was common in luminal/HER2- BC. Using a weighted approach was better than using a mitotically active approach for core to case collapsing. At least a single 1.0-mm core or three 0.6-mm cores are required when designing a study using TMA.

Multimodal analysis of drug transporter expression in gastrointestinal tissue.

OBJECTIVES: Drug transporters affect antiretroviral therapy (ART) tissue disposition, but quantitative measures of drug transporter protein expression across preclinical species are not available. Our objective was to use proteomics to obtain absolute transporter concentrations and assess agreement with corresponding gene and immunometric protein data. DESIGN: In order to make interspecies comparisons, two humanized mouse [hu-HSC-Rag (n = 41); bone marrow-liver-thymus (n = 13)] and one primate [rhesus macaque (nonhuman primate, n = 12)] models were dosed to steady state with combination ART. Ileum and rectum were collected at necropsy and snap frozen for analysis. METHODS: Tissues were analyzed for gene (quantitative PCR) and protein [liquid chromatography-mass spectrometry (LC-MS) proteomics and western blot] expression and localization (immunohistochemistry) of ART efflux and uptake transporters. Drug concentrations were measured by LC-MS/MS. Multivariable regression was used to determine the ability of transporter data to predict tissue ART penetration. RESULTS: Analytical methods did not agree, with different trends observed for gene and protein expression. For example, quantitative PCR analysis showed a two-fold increase in permeability glycoprotein expression in nonhuman primates versus mice; however, proteomics showed a 200-fold difference in the opposite direction. Proteomics results were supported by immunohistochemistry staining showing extensive efflux transporter localization on the luminal surface of these tissues. ART tissue concentration was variable between species, and multivariable regression showed poor predictive power of transporter data. CONCLUSION: Lack of agreement between analytical techniques suggests that resources should be focused on generating downstream measures of protein expression to predict drug exposure. Taken together, these data inform the use of preclinical models for studying ART distribution and the design of targeted therapies for HIV eradication.

Expression of six drug transporters in vaginal, cervical, and colorectal tissues: Implications for drug disposition in HIV prevention.

Effective antiretroviral (ARV)-based HIV prevention strategies require optimizing drug exposure in mucosal tissues; yet factors influencing mucosal tissue disposition remain unknown. We hypothesized drug transporter expression in vaginal, cervical, and colorectal tissues is a contributing factor and selected 3 efflux (ABCB1/MDR1, ABCC2/MRP2, ABCC4/MRP4) and 3 uptake (SLC22A6/OAT1, SLC22A8/OAT3, SLCO1B1/OATP1B1) transporters to further investigate based on their affinity for 2 ARVs central to prevention (tenofovir, maraviroc). Tissue was collected from 98 donors. mRNA and protein expression were quantified using qPCR and immunohistochemistry (IHC). Hundred percent of tissues expressed efflux transporter mRNA. IHC localized them to the epithelium and/or submucosa. Multivariable analysis adjusted for age, smoking, and co-medications revealed significant (P < 0.05) differences in efflux transporter mRNA between tissue types (vaginal ABCB1 3.9-fold > colorectal; vaginal ABCC2 2.9-fold > colorectal; colorectal ABCC4 2.0-fold > cervical). In contrast, uptake transporter mRNA was expressed in <25% of tissues. OAT1 protein was detected in 0% of female genital tissues and in 100% of colorectal tissues, but only in rare epithelial cells. These data support clinical findings of higher maraviroc and tenofovir concentrations in rectal tissue compared to vaginal or cervical tissue after oral dosing. Quantifying mucosal transporter expression and localization can facilitate ARV selection to target these tissues.

Expression of the Pluripotency Transcription Factor OCT4 in the Normal and Aberrant Mammary Gland.

Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor, and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast (from non-pregnant, non-lactating women) showed minimal OCT4 nuclear expression (0.9%). However, this increased in the normal lactating breast (11.4%), with further increase in lactating adenomas, lactating carcinomas, and pregnancy-associated breast cancer (30.7-48.3%). OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs) demonstrated upregulation of OCT4, SOX2, and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both in vitro and in vivo. Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the normal remodeling of the breast toward a milk-secretory organ during pregnancy and lactation. Deregulation of this gene network either within or outside pregnancy and lactation may lead to aberrant breast cell proliferation and malignant transformation, suggesting a role of these genes in both normal lactation and breast oncogenesis.