Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine.

The HIF transcription factor promotes adaptation to hypoxia and stimulates the growth of certain cancers, including triple-negative breast cancer (TNBC). The HIFα subunit is usually prolyl-hydroxylated by EglN family members under normoxic conditions, causing its rapid degradation. We confirmed that TNBC cells secrete glutamate, which we found is both necessary and sufficient for the paracrine induction of HIF1α in such cells under normoxic conditions. Glutamate inhibits the xCT glutamate-cystine antiporter, leading to intracellular cysteine depletion. EglN1, the main HIFα prolyl-hydroxylase, undergoes oxidative self-inactivation in the absence of cysteine both in biochemical assays and in cells, resulting in HIF1α accumulation. Therefore, EglN1 senses both oxygen and cysteine.

MicroRNA-antagonism regulates breast cancer stemness and metastasis via TET-family-dependent chromatin remodeling.

Tumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis in mouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing antimetastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytosine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoter of EMT and breast cancer stemness toward metastasis.

Spatial genomics maps the structure, nature and evolution of cancer clones.

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour1-3. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive4,5. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.

The life history of 21 breast cancers.

Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.

Mutational processes molding the genomes of 21 breast cancers.

All cancers carry somatic mutations. The patterns of mutation in cancer genomes reflect the DNA damage and repair processes to which cancer cells and their precursors have been exposed. To explore these mechanisms further, we generated catalogs of somatic mutation from 21 breast cancers and applied mathematical methods to extract mutational signatures of the underlying processes. Multiple distinct single- and double-nucleotide substitution signatures were discernible. Cancers with BRCA1 or BRCA2 mutations exhibited a characteristic combination of substitution mutation signatures and a distinctive profile of deletions. Complex relationships between somatic mutation prevalence and transcription were detected. A remarkable phenomenon of localized hypermutation, termed "kataegis," was observed. Regions of kataegis differed between cancers but usually colocalized with somatic rearrangements. Base substitutions in these regions were almost exclusively of cytosine at TpC dinucleotides. The mechanisms underlying most of these mutational signatures are unknown. However, a role for the APOBEC family of cytidine deaminases is proposed.

Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-ß and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis.

MicroRNAs are well suited to regulate tumor metastasis because of their capacity to coordinately repress numerous target genes, thereby potentially enabling their intervention at multiple steps of the invasion-metastasis cascade. We identify a microRNA exemplifying these attributes, miR-31, whose expression correlates inversely with metastasis in human breast cancer patients. Overexpression of miR-31 in otherwise-aggressive breast tumor cells suppresses metastasis. We deploy a stable microRNA sponge strategy to inhibit miR-31 in vivo; this allows otherwise-nonaggressive breast cancer cells to metastasize. These phenotypes do not involve confounding influences on primary tumor development and are specifically attributable to miR-31-mediated inhibition of several steps of metastasis, including local invasion, extravasation or initial survival at a distant site, and metastatic colonization. Such pleiotropy is achieved via coordinate repression of a cohort of metastasis-promoting genes, including RhoA. Indeed, RhoA re-expression partially reverses miR-31-imposed metastasis suppression. These findings indicate that miR-31 uses multiple mechanisms to oppose metastasis.

Identifying homologous recombination deficiency in breast cancer: genomic instability score distributions differ among breast cancer subtypes.

PURPOSE: A 3-biomarker homologous recombination deficiency (HRD) score is a key component of a currently FDA-approved companion diagnostic assay to identify HRD in patients with ovarian cancer using a threshold score of ≥ 42, though recent studies have explored the utility of a lower threshold (GIS ≥ 33). The present study evaluated whether the ovarian cancer thresholds may also be appropriate for major breast cancer subtypes by comparing the genomic instability score (GIS) distributions of BRCA1/2-deficient estrogen receptor-positive breast cancer (ER + BC) and triple-negative breast cancer (TNBC) to the GIS distribution of BRCA1/2-deficient ovarian cancer. METHODS: Ovarian cancer and breast cancer (ER + BC and TNBC) tumors from ten study cohorts were sequenced to identify pathogenic BRCA1/2 mutations, and GIS was calculated using a previously described algorithm. Pathologic complete response (pCR) to platinum therapy was evaluated in a subset of TNBC samples. For TNBC, a threshold was set and threshold validity was assessed relative to clinical outcomes. RESULTS: A total of 560 ovarian cancer, 805 ER + BC, and 443 TNBC tumors were included. Compared to ovarian cancer, the GIS distribution of BRCA1/2-deficient samples was shifted lower for ER + BC (p = 0.015), but not TNBC (p = 0.35). In the subset of TNBC samples, univariable logistic regression models revealed that GIS status using thresholds of ≥ 42 and ≥ 33 were significant predictors of response to platinum therapy. CONCLUSIONS: This study demonstrated that the GIS thresholds used for ovarian cancer may also be appropriate for TNBC, but not ER + BC. GIS thresholds in TNBC were validated using clinical response data to platinum therapy.

Police Bias and Low Relatability and Diet Quality: Examining the Importance of Psychosocial Factors in Predominantly Black Communities.

How police bias and low relatability may contribute to poor dietary quality is poorly understood. In this cross-sectional study, we analyzed data from 2021 from a cohort of n = 724 adults living in predominantly Black communities in Pittsburgh, Pennsylvania; these adults were mostly Black (90.6%), low-income (median household income $17,500), and women (79.3%). We estimated direct and indirect paths between police mistrust and dietary quality (measured by Healthy Eating Index (HEI)-2015) through perceived stress, community connectedness, and subjective social status. Dietary quality was poor (mean HEI-2015 score was 50) and mistrust of police was high: 78% of participants either agreed or strongly agreed that something they say might be interpreted as criminal by the police due to their race/ethnicity. Police bias and low relatability was associated with lower perceived social status [Formula: see text]= - 0.03 (95% confidence interval [CI]: - 0.05, - 0.01). Police bias and low relatability was marginally associated with low dietary quality ß = - 0.14 (95% CI: - 0.29, 0.02). Nineteen percent of the total association between police bias and low relatability and lower dietary quality ß = - 0.16 (- 0.01, - 0.31) was explained by an indirect association through lower community connectedness, or how close respondents felt with their community [Formula: see text] Police bias and low relatability may play a role in community connection, social status, and ultimately dietary disparities for Black Americans. Addressing police bias and low relatability is a continuing and pressing public health issue.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Students' consumption of high and low nutrition foods and reduced plate waste by schools' wellness-related policies and garden program participation.

BACKGROUND: Participation in school-based programs that may support children's nutritious dietary behaviours varies across schools. We examined school participation in wellness-related policies, school-based garden programs and students' dietary behaviours. METHODS: Among matching schools who did and did not participate in school-based garden programs, we analysed the lunches of 80 Pittsburgh Public Schools (PPS) students in 1st, 2nd, 6th and 7th grades during Autumn 2019 using digital food photography. We also acquired school wellness policy data. Using cross-sectional linear regression, we estimated the association between school-based garden programming, wellness-related policies and dietary outcomes, adjusting for grade. RESULTS: School's implementation of nutrition services policies was negatively associated with energy wasted from lunch ( ß = - 44.7 , p = 0.01 ${\rm{\beta }}=-44.7,{p}=0.01$ ). The number of semesters the students' school had participated in the garden program was positively associated with students' whole grain consumption ( ß = 0.07 , p < 0.001 ${\rm{\beta }}=0.07,{p}\lt 0.001$ ). CONCLUSIONS: Cross-sectional associations suggest that schools that are more engaged in wellness policies and garden programs may provide environments that are more supportive of students' nutrition than in other schools.

Two for the GOES: Exploring Gambling Outcome Expectancies Scores Across Mixed and Offline-Only Gamblers in Relation to Problem Gambling Risk Status.

As online gambling becomes more prevalent, understanding the motives of online gamblers has become a key focus for research and practice. The aim of this study was to understand differences in gambling-related outcome expectancies between mixed (both online and offline) gamblers and offline-only gamblers, by incorporating gambling harm risk categories from the problem gambling severity index (PGSI). This study comprised a secondary data analysis of the 2015 Northern Territory Gambling Prevalence and Wellbeing Survey. A sample of 1207 individuals in the Northern Territory who had reported gambling at least once in the previous 12 months were used in the analyses. General linear and structural equation modelling were used to ascertain differences in gambling outcome expectancies, in relation to gambling modality (i.e., mixed, offline-only) and PGSI scores. Mixed gamblers tended to score higher on all outcome expectancies than their offline-only counterparts. Outcome expectancy scores were higher in individuals in higher-risk PGSI categories. The escape outcome expectancy was dependent on both modality and risk category. Invariance testing of a low and problem gambling risk subsample revealed differential relationships for both the escape and excitement outcome expectancies for mixed and offline-only gamblers. The results provide an important contribution to the existing literature regarding motivation and outcome expectancies in relation to gambling modality and problem gambling severity. The findings highlight the importance of considering both gambling outcome expectancies and modality when considering problem gambling.

The circular RNome of primary breast cancer.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (R < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of CNOT2, CREBBP, and RERE in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of circCNOT2 was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, CNOT2 levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that circCNOT2 is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.

Improvements in Neighborhood Socioeconomic Conditions May Improve Resident Diet.

Neighborhood socioeconomic conditions (NSECs) are associated with resident diet, but most research has been cross-sectional. We capitalized on a natural experiment in Pittsburgh, Pennsylvania, in which 1 neighborhood experienced substantial investments and a sociodemographically similar neighborhood that did not, to examine pathways from neighborhood investments to changed NSECs and changed dietary behavior. We examined differences between renters and homeowners. Data were from a random sample of households (n = 831) in each of these low-income Pittsburgh neighborhoods that were surveyed in 2011 and 2014. Structural equation modeling tested direct and indirect pathways from neighborhood to resident dietary quality, adjusting for individual-level sociodemographics, with multigroup testing by homeowners versus renters. Neighborhood investments were directly associated with improved dietary quality for renters (ß = 0.27, 95% confidence interval (CI): 0.05, 0.50) and homeowners (ß = 0.51, 95% CI: 0.10, 0.92). Among renters, investments also were associated with dietary quality through a positive association with commercial prices (ß = 0.34, 95% CI: 0.15, 0.54) and a negative association with residential prices (ß = -0.30, 95% CI: -0.59, -0.004). Among homeowners, we did not observe any indirect pathways from investments to dietary quality through tested mediators. Investing in neighborhoods may support resident diet through improvements in neighborhood commercial environments for renters, but mechanisms appear to differ for homeowners.

Homologous recombination deficiency (HRD) score in germline BRCA2- versus ATM-altered prostate cancer.

The homologous recombination deficiency (HRD) score integrates three DNA-based measures of genomic instability, and has been understudied in prostate cancer. Given the recent FDA approval of two PARP inhibitors for prostate cancer, HRD score analysis could help to refine treatment selection. We assessed HRD score (defined as the sum of loss-of-heterozygosity, telomeric allelic imbalance, and large-scale state transitions) in three cohorts of primary prostate cancer, including a Johns Hopkins University (JHU) cohort with germline mutations in BRCA2, ATM, or CHEK2 (n = 64), the TCGA cohort (n = 391), and the PROGENE cohort (n = 102). In the JHU cohort, tumors with germline BRCA2 mutations had higher HRD scores (median = 27) than those with germline ATM or CHEK2 mutations (median = 16.5 [p = 0.029] and 9 [p < 0.001], respectively). For TCGA tumors without underlying HR pathway mutations, the median HRD score was 11, significantly lower than ovarian carcinoma lacking BRCA1/2 mutations (median = 28). In the absence of HR gene mutations, the median HRD score was unexpectedly higher among prostate cancers with TP53 mutations versus those without (17 vs. 11; p = 0.015); this finding was confirmed in the PROGENE cohort (24 vs. 16; p = 0.001). Finally, among eight BRCA2-altered patients who received olaparib, progression-free survival trended longer in those with HRD scores above versus below the median (14.9 vs. 9.9 months). We conclude that HRD scores are low in primary prostate cancer and higher in cases with germline BRCA2 or somatic TP53 mutations. Germline BRCA2-altered cases have significantly higher HRD scores than germline ATM-altered or CHEK2-altered cases, consistent with the lower efficacy of PARP inhibitors among the latter.

Food Insecurity in a Low-Income, Predominantly African American Cohort Following the COVID-19 Pandemic.

Objectives. To examine the impact of COVID-19 shutdowns on food insecurity among a predominantly African American cohort residing in low-income racially isolated neighborhoods.Methods. Residents of 2 low-income African American food desert neighborhoods in Pittsburgh, Pennsylvania, were surveyed from March 23 to May 22, 2020, drawing on a longitudinal cohort (n = 605) previously followed from 2011 to 2018. We examined longitudinal trends in food insecurity from 2011 to 2020 and compared them with national trends. We also assessed use of food assistance in our sample in 2018 versus 2020.Results. From 2018 to 2020, food insecurity increased from 20.7% to 36.9% (t = 7.63; P < .001) after steady declines since 2011. As a result of COVID-19, the United States has experienced a 60% increase in food insecurity, whereas this sample showed a nearly 80% increase, widening a preexisting disparity. Participation in the Supplemental Nutrition Assistance Program (52.2%) and food bank use (35.9%) did not change significantly during the early weeks of the pandemic.Conclusions. Longitudinal data highlight profound inequities that have been exacerbated by COVID-19. Existing policies appear inadequate to address the widening gap.

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ∼25-35 Mya and CFHR1 and CFHR3 ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

Improved Street Walkability, Incivilities, and Esthetics Are Associated with Greater Park Use in Two Low-Income Neighborhoods.

Parks may provide opportunities for people to increase their physical activity and improve health. Yet, parks are generally less plentiful and underutilized in low-income urban neighborhoods compared with more advantaged neighborhoods. Renovations within and around parks may improve park utilization but the empirical evidence supporting this relationship is scarce. This study assessed the impact of greenspace, housing, and commercial investments on street characteristics (walkability, amenities, incivilities/poor esthetics) and park use by examining park use over time in two low-income neighborhoods in Pittsburgh, PA (n = 17 parks), before and after neighborhood-based renovations that were primarily centered in one neighborhood. We used systematic observation of parks, park use, and street blocks surrounding parks to examine the impact of neighborhood changes on park use. We used difference-in-differences to test whether park use and street characteristics surrounding the parks improved more in the intervention neighborhood than in the comparison neighborhood. We also used zero-inflated negative binomial regression with interactions by time to test whether changes in street characteristics were associated with changes in park use over time. We found that improved walkability, incivilities, and esthetics surrounding parks in socioeconomically disadvantaged neighborhoods were associated with greater park use and may help increase visits to underutilized parks.

Broken Windows, Broken Zzs: Poor Housing and Neighborhood Conditions Are Associated with Objective Measures of Sleep Health.

African Americans and socioeconomically disadvantaged individuals have higher rates of a variety of sleep disturbances, including short sleep duration, poor sleep quality, and fragmented sleep. Such sleep disturbances may contribute to pervasive and widening racial and socioeconomic (SES) disparities in health. A growing body of literature demonstrates that over and above individual-level SES, indicators of neighborhood disadvantage are associated with poor sleep. However, there has been scant investigation of the association between sleep and the most proximal environments, the home and residential block. This is the first study to examine the association between objective and self-reported measures of housing and block conditions and sleep. The sample included 634 adults (mean age = 58.7 years; 95% African American) from two low-income urban neighborhoods. Study participants reported whether they experienced problems with any of seven different housing problems (e.g., broken windows) and rated the overall condition of their home. Trained data collectors rated residential block quality. Seven days of wrist actigraphy were used to measure average sleep duration, efficiency, and wakefulness after sleep onset (WASO), and a sleep diary assessed sleep quality. Multivariate regression analyses were conducted for each sleep outcome with housing or block conditions as predictors in separate models. Participants reporting "fair" or "poor" housing conditions had an adjusted average sleep duration that was 15.4 min shorter than that of participants reporting "good" or "excellent" conditions. Those reporting any home distress had 15.9 min shorter sleep and .19 units lower mean sleep quality as compared with participants who did not report home distress. Poor objectively measured block quality was associated with 14.0 min shorter sleep duration, 1.95% lower sleep efficiency, and 10.7 additional minutes of WASO. Adverse housing and proximal neighborhood conditions are independently associated with poor sleep health. Findings highlight the importance of considering strategies that target upstream determinants of sleep health disparities.

An audit tool for longitudinal assessment of the health-related characteristics of urban neighborhoods: implementation methods and reliability results.

BACKGROUND: Improving the neighborhood environment may help address chronic disease and mortality. To identify neighborhood features that are predictors of health, objective assessments of the environment are used. Multiple studies have reported on cross-sectional assessments of health-related neighborhood features using direct observation. As study designs expand to better understand causation and predictors of change, there is a need to test whether direct observation methods are adequate for longitudinal assessment. To our knowledge, this is the first study to report on the reliability of repeated measurements of the neighborhood environment, and their stability, over time. METHODS: The Pittsburgh Hill/Homewood Research on Neighborhood Change and Health (PHRESH) study conducted longitudinal assessments in two low-income, African American neighborhoods at three waves (years 2012, 2015, 2017). The PHRESH audit tool is a modification of earlier validated tools, with an emphasis on environment features relevant for physical activity, sleep, and obesogenic behaviors. Trained data-collector pairs conducted direct observations of a 25% sample of street segments in each neighborhood. At each wave, we audited a sub-sample of street segments twice and assessed reliability using percentage inter-observer agreement and krippendorf's alpha statistics. Stability of these items was assessed as exhibiting moderate or high agreement at every time point. RESULTS: Across waves, a majority (81%) of the items consistently demonstrated moderate to high agreement except for items such as public/communal space, amount of shade, sidewalk features, number of traffic lanes, garden/flower bed/planter, art/statue/monument, amount of trash, and physical disorder. The list of items with poor agreement includes features that are easy to miss (e.g. flower bed/planter), hard to assess from outside (e.g. public/communal space), or may change quickly (e.g. amount of trash). CONCLUSION: In this paper, we have described implementation methods, reliability results and lessons learned to inform future studies of change. We found the use of consistent methods allowed us to conduct reliable, replicable longitudinal assessments of the environment. Items that did not exhibit stability are less useful for detecting real change over time. Overall, the PHRESH direct observation tool is an effective and practical instrument to detect change in the neighborhood environment.