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Background/Objective: Breast cancer incidence in Asian populations has increased in recent years, and variation in prognosis and tumor subtypes indicates that further study is warranted to characterize these differences and identify actionable targets. Patients of Asian ancestry are underrepresented in US registries, and few studies have characterized molecular profiles for these patients. In the current analysis, we assess clinical, pathological, and molecular profiles from self-reported Asian breast cancer patients (AS), in comparison with age-matched Caucasian (CA) and African American patients (AA), to evaluate the influence of Asian ancestry on differential gene expression in breast tumors. Methods: This meta-analysis included cohorts of self-reported AS, CA, and AA with early-stage, invasive breast cancer (EBC) prospectively enrolled in the US from 2011 to 2020 in FLEX (NCT03053193), MINT (NCT01501487), or IMPACt (NCT02670577) trials. AS were significantly younger (mean, 55 years) than CA (mean, 61 years, p ± 0.5 were considered significant. Results: AS tumors were classified as 59% MP HR, compared with 44% HR in age-matched CA (p=0.08) and 64% HR in age-matched AA (p=0.17). AS had a significantly lower rate of obesity (16%, body mass index ≥30) compared with CA (41%) and AA (67%) (p Conclusions: AS were significantly younger and more often pre/peri-menopausal at diagnosis compared with CA and AA, consistent with the literature. Most clinical-pathological factors were similar between age-matched groups, except for the obesity rate, which was significantly lower in AS than in CA or AA. Although not significant, AS had EBC that was more often MP HR than CA and less often HR than AA; studies with larger patient groups will help confirm these trends. The current analysis revealed different underlying gene expression pathways in AS compared with other ethnic groups, which may result in differential clinical outcomes. As genomic profiling data are not widely available for Asian American EBC patients, further analyses are warranted to elucidate these outcomes and identify appropriate therapeutic strategies. Pathology and Genomic Results(unknowns excluded)Asian (n=103)Caucasian (n=103)African American (n=100)p-valueAS vs. CAp-value AS vs. AAER status (IHC)ER Positive69 (94.5%)89 (98.9%)73 (80.2%)0.1250.010ER Negative4 (5.5%)1 (1.1%)18 (19.8%)PR status (IHC)PR Positive64 (87.7%)81 (90%)63 (69.2%)0.4110.006PR Negative9 (12.3%)9 (10%)28 (30.8%)HER2 (IHC/FISH)HER2 Positive3 (4.2%)3 (3.4%)13 (14.3%)0.1720.017HER2 Negative64 (88.9%)85 (95.5%)77 (84.6%)Equivocal5 (6.9%)1 (1.1%)1 (1.1%)MP/BP resultsLuminal A39 (40.6%)51 (53.7%)28 (30.4%)0.2320.043Luminal B45 (46.9%)38 (40.0%)42 (45.7%)HER2 (MP HR)7 (7.3%)4 (4.2%)5 (5.4%)Basal (MP HR)5 (5.2%)2 (2.1%)17 (18.5%) Citation Format: Margaret Chen, Ava Kwong, Carolyn Hendricks, Nina D'Abreo, Laura Lee, Hatem H. Soliman, Charles Cox, Heather M. Kling, Rajith Bhaskaran, Shiyu Wang, Andrea Menicucci, Sarah Untch, William Audeh, FLEX Investigators Group. Molecular profiles and clinical-pathological features of Asian early-stage breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-69.

Background: African American breast cancer patients (AA) are diagnosed at a younger age and present more frequently with triple-negative/Basal tumors than Caucasian American patients (CA). High prevalence of obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome in AA may confound attempts to evaluate the influence of race on gene expression. Previously we showed that differentially expressed genes (DEGs) between AA and CA within the Basal subtype were related to metabolism, translation, and cell signaling pathways (Nunes et al. 2019). However, AA had higher obesity and T2DM rates than CA, and we were unable to distinguish between the influence of metabolic factors and race. In the current analysis, we aim to dissect these factors by comparing clinical and molecular features of Basal-type breast tumors in obese AA and CA. Methods: The prospective, observational FLEX Registry (NCT03053193) includes stage I-III breast cancer patients who receive 70-gene signature (MammaPrint, MP)/80-gene signature (BluePrint, BP) testing and consent to full transcriptome and clinical data collection. This interim substudy included 50 AA and 96 CA (n=146), enrolled from 2017 to present, all obese by body mass index (BMI, ≥30) and whose tumors were MP High Risk and BP Basal subtype. AA were significantly younger (mean, 55 years) than CA (mean, 60 years, p=0.02); thus, an age distribution-matched subset (n=49 AA, n=49 CA) was added for comparison. Gene expression data were quantile normalized using R limma package; DEGs were compared between groups in the following: (1) all AA (n=50) and CA (n=96), (2) AA and 3 random selections of CA (n=50 pairs), and (3) age-matched AA and CA (n=49 pairs). Results: Clinical factors, including tumor stage, nodal stage, and T2DM status were similar between AA and CA, regardless of age-matching. Most tumors were T1/2 (83% AA, 88% CA) and negative for nodal involvement (77% AA, 68% CA). 94% of tumors from AA and 74% of tumors from CA were grade 3 (p=0.17). Notably, 32% of tumors from AA and 46% of tumors from CA were ER+ by immunohistochemistry. Age-matched AA and CA had a 20% rate of T2DM. 152 DEGs were significant (adjusted p Conclusions: Higher prevalence of obesity/T2DM in AA has been proposed as a key factor to explain racial disparities in breast cancer incidence and prognosis, but the current results suggest that race may influence DEGs more than differences in tumor subtype, age, or metabolic factors. This comparison also emphasizes the importance of matched clinical features for DEG analysis and suggests disparities in AA beyond those attributable to clinical differences within the population. DEGs in AA suggest upregulation of Notch-associated aggressiveness, which may be particularly relevant under hypoxic conditions (e.g., obesity), and pathways associated with stemness, metastasis, and chemotherapy resistance. Notch pathway also interacts with key oncogenic pathways, and future studies will reveal the molecular networks underlying racial disparity in AA and CA breast cancer patients. Citation Format: Dipali Sharma, Lisa E. Blumencranz, Heather M. Kling, Sahra Uygun, Sarah Untch, William Audeh, Jennifer A. Crozier, Mehran Habibi, Raquel Nunes. Racial disparities within basal-type breast cancer: Clinical and molecular features of African American and Caucasian obese patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-68.

Background: Breast cancer rates among Asian women are relatively low compared with Western populations; however, rates have increased in recent years, and it is the most common cancer in women. Few studies have characterized clinical-pathological features and molecular subtypes in Asian breast cancer patients, although substantial variation in occurrence among Asian subpopulations has been reported. Here, we report clinical factors, pathology, and molecular profiles of Chinese patients enrolled in the US and Hong Kong, HKSAR, and from age-matched Caucasian (Cau) and African-American (AA) patients. Methods: This analysis included Chinese patients enrolled in the United States (n=24) and Hong Kong (n=42) with early stage, invasive breast cancer for whom clinical characteristics were captured with informed consent, enrolled between 2013 and 2018 (NCT02669745). For ethnicity comparisons, age-matched Cau (n=132) and AA (n=33) patients were included from the prospective, US-based FLEX registry (NCT03053193). Ethnicity was patient reported. Clinical characteristics, pathology, and results from the 70-gene (70-GS, MammaPrint) risk of recurrence and 80-gene (80-GS, BluePrint) molecular subtyping signatures were compared. Results: The median age at diagnosis for Chinese was 51 years, and patients were 53.8% pre-/peri-menopausal. Tumors were predominantly ductal carcinoma, not otherwise specified (NOS) (81.3%), T1 (73.0%), grade 2 (59.7%), estrogen receptor-positive (ER+) (90.6%), and node-negative (82.0%). 70-GS and 80-GS results were available from 64 patients; 56.3% of tumors classified as 70-GS High Risk, and 82.8% Luminal-type, 9.4% HER2-type, and 7.8% Basal-type by 80-GS. Although pooled here for analysis, when examined separately, tumors of Chinese American patients were more frequently HER2-type (5/23; 21.7%) than tumors of Hong Kong patients (1/41, 2.4%) and had some higher risk clinical features, such as greater frequency of grade 3 classification (54.2%). Age-matched Cau patients were similar to Chinese patients in menopausal status (50.4% pre/peri-menopausal); age-matched AA patients were predominantly (58.6%) post-menopausal. Tumors of Cau patients were predominantly ductal carcinoma NOS (87.9%), T1 (73.4%), grade 2 (51.6%), node-negative (82.4%), ER+ (89.2%), 70-GS Low Risk (52.3%) and 80-GS Luminal-type (86.4%). Tumors of AA patients were predominantly ductal carcinoma, not otherwise specified (NOS) (86.7%), T1 (50.0%), grade 3 (53.6%), node-negative (79.3%), ER+ (72.7%), 70-GS High Risk (75.8%), and 80-GS Luminal-type (60.6%), although a substantial portion were 80-GS Basal-type (33.3%). Conclusions: The current study revealed some disparities in clinical and molecular features of breast tumors between Chinese American and Hong Kong patients, and among Chinese, Cau, and AA patients. Some features typically associated with higher clinical risk were more prevalent in Chinese American patients than in Hong Kong patients, suggesting a possible interaction between genetics and lifestyle factors, and perhaps influenced by immigration. However, given the small sample size, these data should be interpreted with caution, and further study is needed to validate these trends. Future studies of breast cancer in ethnic subpopulations should incorporate evaluation of genomic profiling. BluePrint Molecular Subtyping ResultsPatients Luminal-typeHER2-typeBasal-typeTotalChinese (pooled)53 (82.8%)6 (9.4%)5 (7.8%)64Hong Kong37 (90.2%)1 (2.4%)3 (7.3%)41US16 (69.6%)5 (21.7%)2 (8.7%)23Caucasian (US)114 (86.4%)6 (4.6%)12 (9.1%)132African-American (US)20 (60.6%)2 (6.1%)11 (33.3%)33 Citation Format: Ava Kwong, Sarah Untch, Heather M. Kling, Christine Finn, William Audeh, Vivian Shin, Sherene Ishtihar, Margaret Chen. Different MammaPrint and BluePrint molecular profiles and clinical-pathological features of early stage breast cancer in Chinese patients in the United States and Hong Kong [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-10-15.

Background discussion: Genomic signatures are revolutionizing the definition, identification, and treatment of breast cancer. To precisely stratify breast cancers into actionable subgroups, full genome (FG) expression data and matching clinical data must be aggregated into a large, real-world dataset. Such a dataset will accelerate research and discovery, especially for smaller patient subsets that are not as widely represented within the current body of literature. Trial design and aims: FLEX is a multicenter, prospective, population-based, observational trial for patients with Stage I, II, and III breast cancer. All patients with stage I to III breast cancer who receive MammaPrint (MP), with or without BluePrint (BP) on a primary breast tumor are eligible for enrollment. The study’s primary aim is to create a large scale, population-based registry of full genome expression data matched with clinical data to investigate new gene associations with prognostic and/or predictive value in a real-world setting. Secondary objectives include utilizing the shared study infrastructure to examine and generate hypotheses for targeted subset analyses and/or trials based on full genome expression data. The design of FLEX allows targeted sub-studies and sub-analyses to be added as appendices after the initial baseline study is opened. Data collection follows patients from diagnosis through 10 years of follow-up. Patients enrolled in the initial study are also eligible for inclusion in sub-studies for which they meet all criteria and additional consent is not required. Additional clinical data will be collected as specified in the appendix protocols. The FLEX collaborative platform allows participating investigators the opportunity to author their own sub-study protocols, as approved by the FLEX Steering Committee of their peers. 14 sub-studies have already been identified and approved (Table 1). Eligibility and accrual: The study will enroll a minimum of 10000 patients aged ≥18 years with histologically proven invasive stage I, II, or III breast cancer. Since April 2017 over 2362 patients have been enrolled. Trial contact information: NCT03053193 FLEX@agendia.com TABLE 1PIINSTITUTIONTYPEADDITIONAL CONSENTARM(S)DESCRIPTIONMehran HabibiJohns HopkinsLocal SubstudyYesNeoadjuvantComprehensive gene expression profiling of breast cancer in patients receiving short-course endocrine therapy prior to surgeryMehran HabibiJohns HopkinsLocal SubstudyYesAllCorrelation of the microbiome with breast cancer gene expressionThomas LomisValley Breast ClinicCollaborative group substudyYesNeoadjuvantComplementary data collection for patients participating in the ODM-201 trial. FLEX will provide gene expression profiling for exploratory and signature discovery purposesIan GradyNorth Valley Breast CareSubstudyNoAllImpact of genomic risk classification on travel time to receive breast cancer care: a QOL studyPat WhitworthNashville Breast CenterSubstudyNoNeoadjuvantGenomic reclassification of large tumors in patients eligible to receive neoadjuvant therapyAdam BrufskyUniversity of Pittsburgh Medical CenterSubstudyNoNeoadjuvant, adjuvantResponse to standard chemotherapy regimens in clinically ER+/PR+/HER2+ (triple positive) patients according to BluePrint molecular subtypesAdam BrufskyUniversity of Pittsburgh Medical CenterSubstudyNoAllExpression signatures by response to bisphosphonates in ER+ patients receiving adjuvant therapy (SOC option), or for osteoporosis after primary treatmentAdam BrufskyUniversity of Pittsburgh Medical CenterSubstudyNoAllGene expression in breast cancer patients with obesityJennifer A. CrozierBaptist MD Anderson Cancer CenterSubstudyNoAllMP and BP in male breast cancerJennifer A. CrozierBaptist MD Anderson Cancer CenterSubstudyNoAllMP and BP evaluation in breast cancer patients ≥70Jennifer A. CrozierBaptist MD Anderson Cancer CenterSubstudyNoAllFG evaluation in invasive lobular carcinomaJennifer A. CrozierBaptist MD Anderson Cancer CenterSubstudyNoAllMP and BP relation to clinical PR positivityJennifer A. CrozierBaptist MD Anderson Cancer CenterSubstudyNoAllMP and BP relation to clinical Ki67 scoreJennifer A. CrozierBaptist MD Anderson Cancer CenterSubstudyNoAllMP and BP in metaplastic breast cancer Citation Format: Jennifer A Crozier, Gordan Srkalovic, Adam Brufsky, Mehran Habibi, Pat Whitworth, Charles Cox, Ian Grady, Thomas Lomis, Nina D'Abreo, Erin B Yoder, Sarah Untch, Heather Kling, William Audeh, Bastiaan Van der Baan, FLEX Investigators Group. The FLEX real world data platform explores new gene expression profiles and investigator-initiated protocols in early stage breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-17-02.

Background: Breast cancer (BC) mortality is higher in African-American women (AA) than in Caucasian women (CA). AA are also diagnosed at a younger age, have more aggressive subtypes and greater incidence of metabolic dysfunction such as obesity and diabetes. These disparities have been attributed to a confluence of socioeconomic, genetic and epigenetic factors. However, the distinctive tumor biology of AA BC is not yet fully elucidated, as AA remain underrepresented in breast cancer studies and databases. Here, we compared clinical and molecular BC features of AA and CA patients for insights into mechanisms associated with these racial disparities. Methods: The FLEX Registry Trial (NCT03053193) is a prospective study evaluating tissue collected from patients with stage I-III BC who have consented to receive MammaPrint(MP)/BluePrint(BP) and clinically annotated full genome (FG) data. FLEX subset analyses investigate new gene associations that may be relevant to BC biology. This sub-study includes 160 AA and 199 CA patients (n=359) enrolled since April 2017. Clinical characteristics used in the analysis include menopausal status, metabolic factors, stage, grade and IHC results. A comprehensive publication search (PubMed) was conducted to validate candidate genes involved in BC in AA, BC genes associated with epigenetic regulation and genes associated with metabolic syndrome. Hierarchical clustering was performed on FG microarray (Agilent) intensity data focusing on the candidate genes (50 probes targeting 37 unique genes). Gene expression was compared between race and MP/BP risk groups. Results: AA were predominately MP High Risk (HR) 67.5%, BP Luminal B 40.2%, BP Basal 22.6%. Interestingly, 40.0% of AA BP Basal were classified by IHC as ER+. CA were MP Low Risk (LR) 54.8%, BP Luminal A 54.8%. Clinically, there were no differences in histology, tumor size, nodal or menopausal status between AA and CA patients. AA had higher grade tumors, higher rates of type 2 diabetes and obesity. Three comparisons were performed: 1) AA and CA, irrespective of MP result, 2) AA and CA HR MP only, and 3) AA only, irrespective of MP result. In comparison 1) 15 unique genes showed significant differences (p Conclusions: Here we show AA patients were enriched for genes associated with metabolic syndrome and epigenetic regulation of metabolic syndrome. The aberrant function of these genes has been implicated in tumorigenesis, dysregulation of metabolism and drug resistance. Further validation is warranted to fully understand the association of these genes with the unique biology of breast cancer in AA. Citation Format: Raquel Nunes, Lisa E. Blumencranz, Heather M Kling, Sahra Uygun, Sarah Untch, Erin B Yoder, Jennifer A Crozier, William Audeh. Racial disparities in breast cancer: Identifying predisposing clinical and molecular features associated with African American patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-10-08.

Background Increased usage of genomic risk assessment assays suggests increased reliance on data provided by these assays to guide therapy decisions. The current study aimed to assess the change in treatment decision and physician confidence based on the 70-gene risk of recurrence signature (70-GS, MammaPrint) and the 80-gene molecular subtype signature (80-GS, BluePrint) in early stage breast cancer patients. Methods IMPACt, a prospective, case-only study, enrolled 452 patients between November 2015 and August 2017. The primary objective population included 358 patients with stage I-II, hormone receptor-positive, HER2-negative breast cancer. The recommended treatment plan and physician confidence were captured before and after receiving results for 70-GS and 80-GS. Treatment was started after obtaining results. The distribution of 70-GS High Risk (HR) and Low Risk (LR) patients was evaluated, in addition to the distribution of 80-GS compared to IHC status. Results The 70-GS classified 62.5% (n = 224/358) of patients as LR and 37.5% (n = 134/358) as HR. Treatment decisions were changed for 24.0% (n = 86/358) of patients after receiving 70-GS and 80-GS results. Of the LR patients initially prescribed CT, 71.0% (44/62) had CT removed from their treatment recommendation. Of the HR patients not initially prescribed CT, 65.1% (41/63) had CT added. After receiving 70-GS results, CT was included in 83.6% (n = 112/134) of 70-GS HR patient treatment plans, and 91.5% (n = 205/224) of 70-GS LR patient treatment plans did not include CT. For patients who disagreed with the treatment recommended by their physicians, most (94.1%, n = 16/17) elected not to receive CT when it was recommended. For patients whose physician-recommended treatment plan was discordant with 70-GS results, discordance was significantly associated with age and lymph node status. Conclusions The IMPACt trial showed that treatment plans were 88.5% (n = 317/358) in agreement with 70-GS results, indicating that physicians make treatment decisions in clinical practice based on the 70-GS result. In clinically high risk, 70-GS Low Risk patients, there was a 60.0% reduction in treatment recommendations that include CT. Additionally, physicians reported having greater confidence in treatment decisions for their patients in 72% (n = 258/358) of cases after receiving 70-GS results. Trial registration “Measuring the Impact of MammaPrint on Adjuvant and Neoadjuvant Treatment in Breast Cancer Patients: A Prospective Registry” (NCT02670577) retrospectively registered on Jan 27, 2016.

We investigated the relationship of monocytes, alveolar, and tissue-resident macrophage populations and the development of pulmonary arterial hypertension (PAH) in a nonhuman primate model of HIV infection. A prospective study of simian immunodeficiency virus-associated pulmonary arterial hypertension (SIV-PAH) was done. Rhesus macaques (n = 21) were infected with SIV. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were analyzed for monocyte and macrophage phenotypes and inflammatory mediators. Serial right heart catheterizations were performed at three time points throughout the study to assess hemodynamic alterations and the development of PAH. All 21 animals showed similar courses of SIV infection with an increasing proinflammatory plasma environment. At 6 months postinfection (mpi), 11 of 21 animals developed SIV-PAH (mPAP ≤25 mmHg; right ventricular systolic pressure [RVSP] ≤36 mmHg). PAH+ animals had an increased frequency of proinflammatory, nonclassical monocytes (CD14dimCD16+) (p = .06) in the peripheral blood and CD14+CCR7-CD163-CD206+ macrophages (p = .04) in BALF compared with PAH- animals at 6 mpi. Increased frequencies of these monocyte and macrophage phenotypes correlated with elevated RVSP (p = .04; p = .03). In addition, PAH+ animals had greater frequencies of tissue resident inflammatory M1-like CD68+STAT1+ (p = .001) and M2a-like CD68+STAT3+ macrophages (p = .003) and a lower frequency of anti-inflammatory M2c-like CD68+STAT6+ macrophages (p = .003) as well as fewer interleukin (IL)-10+ cells (p = .01). The results suggest that HIV-PAH is associated with skewing of monocytes and alveolar macrophages toward a proinflammatory, profibrotic phenotype. Furthermore, PAH+ animals may have diminished capacity to downregulate exaggerated chronic inflammation, as indicated by lower levels of IL-10 in PAH+ animals, contributing to disease progression.

Pulmonary arterial hypertension (PAH) is a life-threatening disease with higher incidence in HIV-infected compared with noninfected patients. SIV-infected NHP develop clinical manifestations of HIV infection, including PAH. To understand the pathogenesis of PAH and determine the relationship between hemodynamic changes and clinical characteristics associated with SIV infection, we performed right heart catheterization and echocardiographic imaging of 21 rhesus macaques before and after SIV infection. Between 6 and 12 mo after infection, 11 of the 21 animals had elevated mean pulmonary arterial pressure (mPAP; greater than 25 mm Hg). RV involvement was evident as increased RV glucose uptake in PAH(+) macaques on positron emission tomography–coupled CT compared with uninfected animals. RV and pulmonary vascular collagen deposition were elevated in PAH(+) animals. At 12 mo after infection, 6 of the 21 macaques (28.6%) exhibited continued increase in mPAP (progressive PAH), whereas 5 animals (23.8%) had reduced pressure (transient PAH). SIV infection of rhesus macaques led to 3 distinct outcomes with regard to hemodynamic function. Hemodynamic alterations correlated with specific inflammatory profiles and increased RV and pulmonary arterial fibrosis but not with viral load, sex, or CD4(+) T-cell levels. This model of a natural cause of PAH provides insight into disease pathways that are important for the development of novel therapeutic targets.

Pulmonary arterial hypertension (PAH) is a life-threatening disease with higher incidence in HIV-infected compared with noninfected patients. SIV-infected NHP develop clinical manifestations of HIV infection, including PAH. To understand the pathogenesis of PAH and determine the relationship between hemodynamic changes and clinical characteristics associated with SIV infection, we performed right heart catheterization and echocardiographic imaging of 21 rhesus macaques before and after SIV infection. Between 6 and 12 mo after infection, 11 of the 21 animals had elevated mean pulmonary arterial pressure (mPAP; greater than 25 mm Hg). RV involvement was evident as increased RV glucose uptake in PAH+ macaques on positron emission tomography-coupled CT compared with uninfected animals. RV and pulmonary vascular collagen deposition were elevated in PAH+ animals. At 12 mo after infection, 6 of the 21 macaques (28.6%) exhibited continued increase in mPAP (progressive PAH), whereas 5 animals (23.8%) had reduced pressure (transient PAH). SIV infection of rhesus macaques led to 3 distinct outcomes with regard to hemodynamic function. Hemodynamic alterations correlated with specific inflammatory profiles and increased RV and pulmonary arterial fibrosis but not with viral load, sex, or CD4+ T-cell levels. This model of a natural cause of PAH provides insight into disease pathways that are important for the development of novel therapeutic targets.

1009 Background: African American breast cancer patients (AA) are diagnosed younger, have more high-risk features, and poorer clinical outcomes than non-Hispanic White patients (NHW), despite similar treatments. Although comorbidities such as obesity and metabolic syndrome may contribute to differences, ancestry-specific factors and effects of structural violence that disproportionately afflict AA individuals may influence tumor biology and outcomes. We previously reported differentially expressed genes (DEGs) associated with tumor aggressiveness in Basal tumors from AA compared with NHW (Sharma et al., 2020). Here, we compare DEGs in luminal tumors between AA and NHW. Methods: The prospective, observational FLEX study (NCT03053193) includes stage I-III breast cancer patients who receive 70-gene signature (MammaPrint/MP)/80-gene signature (BluePrint/BP) testing and consent to full transcriptome and clinical data collection. AA (n=364) and NHW (n=400, random selection) with BP luminal tumors, enrolled from 2017 to present, were included. Race/ethnicity was self-reported. AA were younger than NHW (mean, 59 vs. 62 years, p=0.001); thus, an age-matched subset (n= 360 AA, NHW) was compared. Differential gene expression analysis was performed with R limma package. Comparisons were made between AA and age-matched or randomly selected NHW in: (1) all, (2) luminal A, (3) luminal B, and (4) luminal B, obese. DEGs with FDR1.7) in the luminal B age-matched comparison, 2 ( PSPH and LINC01139) were also found in the luminal B, obese subset. Consistently upregulated DEGs in AA were associated with metabolism, translation, and cellular stress response pathways. Conclusions: We found significant transcriptomic differences between luminal tumors from AA and NHW, when controlling for age, obesity, and genomic classification. A subset of DEGs in luminal B tumors were consistent with those in Basal tumors, suggesting that similar race-associated factors drive DEGs regardless of tumor subtype. DEGs that may be unique to AA luminal tumors were also found. This study suggests that some biological differences in breast tumors may result from patient ancestry or shared adverse socioeconomic exposures and underscores the need for inclusion of diverse patient groups in clinical trials. Clinical trial information: NCT03053193.

e12568 Background: Genomic risk of recurrence assays provide prognostic information that aids treatment recommendations for patients with early breast cancer (EBC). The 70-gene signature (MammaPrint/MP) and the 21-gene assay (Oncotype DX/ODX) were both validated by large prospective, randomized clinical trials. Previous studies have directly compared the performance of these assays, with moderate discordance widely observed. Recent work showed poor prognostic performance of the ODX in African American (AA) EBC patients (Hoskins et al., 2021). Here, we compare the concordance of two genomic tests in an AA cohort. Methods: This retrospective analysis included a subset of self-reported AA patients from the prospective, observational PROMIS (n = 66, NCT01617954) and FLEX (n = 29, NCT03053193) registry trials, for whom both MP and ODX RS results were available. Patients were enrolled from 2012 to present. The PROMIS trial enrolled patients with intermediate ODX RS (18-30). MP results were obtained from standard testing performed centrally by Agendia, Inc. (Irvine, CA); ODX testing was performed by Genomic Health, Inc. (Redwood City, CA) and results were collected from patient case report forms. Results: AA patients (n = 95) included in this sub-analysis were 77% post-menopausal with a median age of 60 years. 28% of patients were aged ≤ 50 years. Of patients with metabolic data (n = 49), 63% were obese by body mass index (BMI > 30), and 23% had type 2 diabetes mellitus (T2DM). Tumors were predominantly invasive ductal carcinoma (82%), T1 (73%), grade 2 (53%), ER+ (99%), HER2-negative (94%), lymph node-negative (86%), and MP High Risk (66%). By ODX clinical RS, 17% of tumors classified as low (0-17), 80% intermediate (18-30), and 3% high (31-100) risk. As expected, the large intermediate RS group reflects the inclusion of PROMIS patients. By TAILORx RS ranges, 76% of tumors were low risk (≤25). Of these, 61% (n = 44/72) classified as MP High Risk. Notably, 62% (n = 41/66) of tumors with TAILORx intermediate RS (11-25) were MP High Risk. Conclusions: The overall discordance between MP and ODX was 51% in these AA patients. Discordance was more frequent (61%) in ODX low risk (RS≤25) cases. Notably, of tumors with TAILORx intermediate RS (11-25), the majority (62%) classified as MP High Risk. Combined with previously published data in AA patients (Nunes et al., 2016), this results in a total of 57% (n = 52/91) of ODX low RS (≤25) tumors classified as MP High Risk. Recent data indicate that AA patients who receive a low (≤10 or ≤25) or intermediate RS (11-25) have higher recurrence rates and lower survival than White/Caucasian EBC patients with the same RS. In the current study, clinical outcomes are forthcoming; however, these data suggest the majority of AA patients are more likely to receive a MP High Risk result, which may capture the diversity of pathways driving tumor metastasis. Clinical trial information: NCT01617954, NCT03053193.

559 Background: Lymphovascular invasion (LVI), the passage of carcinoma cells through lymphatic and blood vessels, is an important early step in metastasis; however, LVI is excluded from most breast cancer (BC) clinical risk assessments. Previous studies assessed the prognostic value of LVI to estimate clinical outcomes. To gain understanding of the molecular basis of LVI, we evaluated differentially expressed genes (DEGs) between tumors with LVI versus those without LVI, stratified by the 70-gene signature (MammaPrint/MP) and 80-gene molecular subtyping signature (BluePrint/BP). Methods: The prospective, observational FLEX Study (NCT03053193) includes stage I-III BC patients who receive MP/BP testing and consent to full transcriptome and clinical data collection. Patients with LVI (n=581) and without LVI (n=600, randomly selected), enrolled from 2017 to present, were included. LVI was assessed by local pathology laboratories. Differential gene expression analysis of 44k Agilent microarray data was performed with R limma package. DEGs were compared within all samples, BP Luminal subtype, MP risk groups (Low Risk [LR]/Luminal A and High Risk [HR]/Luminal B), and by lymph node (LN) status. DEGs with FDR

Background: Invasive lobular carcinoma (ILC) comprises 10-15% of breast tumors and is the second most common histological type after invasive ductal carcinoma (IDC). Patients with ILC are often diagnosed at an older age and more advanced stage than those with IDC. Late recurrences and worse long-term survival suggest the need for improved approaches to treatment optimization and exploration of molecular pathways unique to ILC. Although previous reports have described comprehensive transcriptomic profiling of ILC, these were limited by small sample sizes. Furthermore, differential gene expression between ILC and IDC within genomic risk groups and molecular subtypes has yet to be explored. Here we characterize differential gene expression between ILC and IDC in a large, age-matched patient subset categorized by 70-gene signature/MammaPrint (MP) risk and 80-gene signature/BluePrint (BP) subtype. Methods: The prospective FLEX Registry (NCT03053193) includes stage I-III primary invasive breast cancer patients who receive MP/BP testing and consent to full transcriptome and clinical data collection. This sub-analysis included 450 ILC patients enrolled from 2017 to present. Compared with a random selection of IDC patients (n=450, mean age, 60 years), ILC patients were older (mean, 63 years, p ± 1.0 were considered significant. Results: ILC represented 13% of FLEX cases (n=450/3562), and were 81% lymph node-negative, 99% ER+, 94% HER2-negative, and 68% MP Low Risk (LR). By BP, ILC were 99% Luminal, 1% HER2, and Conclusions: Here we report differential clinical and molecular characteristics between ILC and IDC in a large, age-matched patient subset. Regardless of MP risk, expression of CDH1 was lower in ILC compared with IDC. Approximately one-third of ILCs were MP HR, and we report a greater number and diversity of DEGs between HR ILC and HR IDC compared with LR tumors, in particular genes related to TGFβ signaling. TGFβ pathway genes play a variety of roles in the tumor microenvironment, including induction of angiogenesis, fibroblast growth factor stimulation, and inhibition and/or exclusion of an immune response. These results suggest that therapeutic strategies targeting the TGFβ pathway may be future avenues of exploration in ILC, although further studies are warranted to characterize underlying molecular mechanisms. Citation Format: Beth-Ann Lesnikoski, Jennifer A. Crozier, Gordan Srkalovic, Patricia Robinson, Clodia Osipo, Kaylan Banda, Heather M. Kling, Josien Haan, William Audeh, FLEX Investigators Group. Differential gene expression in luminal-type invasive lobular carcinoma and invasive ductal carcinoma by MammaPrint risk stratification [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-03.

591 Background: Although advances in immunotherapy for the treatment of breast cancer have been minimal compared with other cancers, studies demonstrating tumor-infiltrating lymphocytes and immunomodulatory gene activation in the tumor microenvironment suggest the importance of antitumor immune responses in clinical outcomes. A 12-chemokine gene score has been shown to predict the presence of ectopic lymph node-like structures (ELN) in the tumor microenvironment and improved survival in melanoma, colon cancer, and breast cancer patients (Prabhakaran, 2017). Here, we evaluated this signature in an independent dataset of breast cancer patients treated with neoadjuvant chemotherapy. Methods: Tumor specimens used in this retrospective analysis (n = 92) were from breast cancer patients enrolled in either MINT (NCT0151487) or NBRST (NCT01479101) neoadjuvant registry trials from 2011 to 2016. Clinical data were captured with informed consent, and 70-gene signature (70-GS), 80-gene signature (80-GS), and full transcriptome data were generated by Agendia, Inc. Gene expression data were quantile normalized using R limma package. Principal component analysis (PCA) was performed on the normalized dataset using R princomp package. Chemokine score (CS) was defined as the first principal component values resulting from PCA. 70-GS/80-GS and clinical data were evaluated in relation to CS. CS were compared using Mann-Whitney test. Results: Of 92 breast tumors available for analysis, 84% were 70-GS High Risk (HR). Tumors were 39% Luminal-type, 24% HER2-type, and 32% Basal-type by 80-GS. HR tumors had higher CS than 70-GS Low Risk (LR) tumors (p < 0.001). 80-GS Basal-type, HER2-type, and Luminal B tumors had higher CS than Luminal A tumors (p < 0.01 for each comparison). High grade and ER-negative tumors seemed to have a high CS, although not significantly. Tumors from patients who achieved a pathological complete response (pCR) following neoadjuvant chemotherapy had higher CS than patients with residual cancer burden (p = 0.048). Conclusions: The current study demonstrated a significantly higher CS in 70-GS HR tumors and those which achieved pCR following neoadjuvant chemotherapy. Although further study is needed to evaluate the association of high CS with tumor-associated ELN, these results support previous work demonstrating that, although high CS is associated with aggressive clinical features, it also predicts superior clinical outcomes. The current study suggests validation of the 12-chemokine gene score in an independent dataset of breast cancer patients.

TPS7088 Background: Genomic expression profiles have enabled the classification of breast cancers into molecular sub-types and provide prognostic information about the metastatic potential of the tumor, both of which have implications for the personalized treatment of breast cancer beyond clinical and pathological features. However, to precisely stratify tumors into actionable subgroups, full genome expression data should be combined with comprehensive clinical information. The FLEX Registry aims to aggregate a large, real-world dataset, which will enable discovery of novel genomic profiles, particularly for patient subsets that are underrepresented in traditional clinical trials and will contribute to improved precision in the management of breast cancer. Methods: The FLEX Registry (NCT03053193) is a multi-center, prospective, observational trial for patients with Stage I, II, and III breast cancer. Patients with stage I-III breast cancer who receive the 70-gene signature risk of recurrence test, with or without the 80-gene signature molecular sub-typing test, on a primary tumor are eligible for enrollment. The primary objective of FLEX is to create a large scale, population-based registry that links complete clinical data with full genome expression data to elucidate new prognostic and/or predictive gene associations in a real-world setting. The FLEX Registry employs a shared study infrastructure to develop and investigate hypotheses for targeted subset analyses and/or clinical trials based on full genome expression data. The adaptable protocol is designed to be amended with the inclusion of targeted sub-studies. Patients enrolled in the initial study are eligible for inclusion in sub-studies for which they meet all eligibility criteria and additional consent is not required. Data will be collected on patients from diagnosis through 10 years of follow-up and any necessary additional clinical data will be collected as specified in the appendix protocols. Target enrollment is a minimum of 10,000 patients; >4,000 patients have enrolled since April 2017 at more than 80 sites, including seven National Cancer Institute-designated comprehensive cancer centers. The FLEX collaborative platform enables participating investigators the opportunity to author their own sub-study protocols, as approved by the FLEX Steering Committee. Fifteen sub-studies have been approved for investigation within the FLEX Registry. Clinical trial information: NCT03053193 .

e19291 Background: Invasive lobular carcinoma (ILC), the second most common histological breast cancer type, comprises 10-15% of breast tumors. Although ILC is thought to have a low risk of relapse, patient survival data vary. ILC treatment guidelines mirror those for invasive ductal carcinoma (IDC); however, these were extrapolated from trials that included predominantly IDC. The FLEX Registry captures data from real-world breast cancer patients; the current sub-study evaluated molecular profiles and treatment recommendations in ILC. Methods: The FLEX Registry (NCT03053193) includes stage I-III primary invasive breast cancer patients who receive 70-gene signature (70-GS)/80-gene signature (80-GS) testing and consent to full transcriptome and clinical data collection. This sub-analysis includes 335 ILC patients and 2,179 IDC patients enrolled from 2017 to present. 70-GS stratified tumors by risk of distant metastasis [High (HR), Low (LR), Ultralow (UL) Risk], and 80-GS classified tumors by molecular subtype (Luminal, HER2, or Basal type). Genomic and clinical data were compared for ILC and IDC using chi-square or Fisher’s exact test. Results: ILC represented 13% of FLEX cases (n = 335/2752); 81% were lymph node-negative, 99% ER-positive, and 94% HER2-negative. Clinical risk assessment (MINDACT criteria) classified 61% of ILC and 53% of IDC as low risk (p = 0.03). 70-GS risk distribution in ILC (18% UL, 51% LR, 31% HR) differed from IDC (13% UL, 34% LR, 53% HR; p < 0.01). Discordance between 70-GS and clinical risk was greater in ILC (43%) than IDC (30%, p < 0.01). 80-GS results also differed between ILC (98% Luminal, 1.6% HER2, 0.3% Basal) and IDC (85% Luminal, 3% HER2, 11% Basal; p < 0.01). Inclusion of chemotherapy (CT) in treatment plans was associated with 70-GS risk (p < 0.01); treatment plans that disagreed with 70-GS results were associated with discordant clinical risk (p < 0.05). Treatment plans disagreed with 70-GS in 15% of ILC and 12% of IDC total cases (p = 0.08), and more frequently in HR cases (25% in ILC, 14% in IDC; p < 0.01). Conclusions: The FLEX Registry includes patients with ILC consistent with real-world breast cancer frequencies. ILC demonstrated genomic risk and subtype profiles distinct from IDC. Treatment plans largely agreed with 70-GS results; however, discordance was more frequent in HR ILC than IDC. Future studies will evaluate clinical outcomes; however, these results demonstrate the added value of molecular profiling in ILC and the utility of real-world registry data in evaluating uncommon breast tumor types. Clinical trial information: NCT03053193 .

Pneumocystis (Pc) is a ubiquitious fungal pathogen that causes pneumonia (PCP) and Pc-related pulmonary sequelae in HIV-infected individuals and other immunocompromised populations. Deficiencies in CD4 T cell populations and immunologic dysfunction can limit humoral immunity and vaccine efficacy in Pc-susceptible populations. The ultimate goal of the field is to develop an effective vaccine that can overcome immune deficits in at risk populations and induce long-lasting protective immunity to Pc. Toward this goals, our laboratory has established a model of PCP co-infection in simian immunodeficiency virus (SIV)-infected non-human primates (NHP). We have previously identified a recombinant protein sub-unit vaccine, KEX1, that induces robust anti-Pc immunity in immune-competent macaques that is durable and prevents Pc pneumonia following simian immunodeficiency virus (SIV)-induced immunosuppression and subsequent Pc challenge (Kling and Norris, 2016). In the present study, we used the SIV model of HIV infection to address whether therapeutic vaccination with KEX1 can be effective following virus-induced immunosuppression. Type I, or invariant natural killer T (iNKT) cells have the potential to provide CD4-independent B cell help. When activated, iNKT cells are capable of rapidly expressing a wide array of cytokines and co-stimulatory molecules that can enhance humoral responses. The central hypothesis of this study is that a therapeutic vaccination strategy can induce invariant natural killer (iNKT) cell-B cell help to boost anti-KEX1 titers and enhance immunity to Pc during virus-induced immunosuppression.

Pulmonary diseases and infections are among the top contributors to human morbidity and mortality worldwide, and despite the successful history of vaccines and antimicrobial therapeutics, infectious disease still presents a significant threat to human health. Effective vaccines are frequently unavailable in developing countries, and successful vaccines have yet to be developed for major global maladies, such as tuberculosis. Furthermore, antibiotic resistance poses a growing threat to human health. The "Challenges and Future in Vaccines, Drug Development, and Immunomodulatory Therapy" session of the 2013 Pittsburgh International Lung Conference highlighted several recent and current studies related to treatment and prevention of antibiotic-resistant bacterial infections, highly pathogenic influenza, respiratory syncytial virus, and tuberculosis. Research presented here focused on novel antimicrobial therapies, new vaccines that are either in development or currently in clinical trials, and the potential for immunomodulatory therapies. These studies are making important contributions to the areas of microbiology, virology, and immunology related to pulmonary diseases and infections and are paving the way for improvements in the efficacy of vaccines and antimicrobials.

BACKGROUND Despite antiretroviral therapy and trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis, Pneumocystis pneumonia remains an important serious opportunistic infection in HIV-infected persons. Pneumocystis (Pc) colonization in HIV-infected individuals and in HIV-uninfected smokers is associated with chronic obstructive pulmonary disease (COPD). We previously developed a nonhuman primate model of HIV infection and Pc colonization and demonstrated that Pc colonization correlated with COPD development. In the present study, we examined kinetics of COPD development in non-human primate and tested the effect of Pc burden reduction on pulmonary function by TMP-SMX treatment. METHODS Cynomolgus macaques (n = 16) were infected with simian/human immunodeficiency virus (SHIV89.6P), and natural Pc colonization was examined by nested polymerase chain reaction of serial bronchoalveolar lavage fluid and anti-Pc serology. RESULTS Eleven of 16 monkeys became Pc colonized by 16 weeks post simian-human immunodeficiency virus (SHIV) infection. Pc colonization of SHIV-infected monkeys led to progressive declines in pulmonary function as early as 4 weeks after Pc detection. SHIV-infected and Pc-negative monkeys maintained normal lung function. At 25 weeks post-SHIV infection, TMP-SMX treatment was initiated in 7 Pc-positive (Pc+) (TMP: 20 mg/kg and SMX: 100 mg/kg, daily for 48 weeks) and 5 Pc-negative (Pc-) monkeys. Four SHIV+/Pc+ remained untreated for the duration of the experiment. Detection frequency of Pc in serial bronchoalveolar lavage fluid (P < 0.001), as well as plasma Pc antibody titers (P = 0.02) were significantly reduced in TMP-SMX-treated macaques compared with untreated. CONCLUSIONS Reduction of Pc colonization by TMP-SMX treatment did not improve pulmonary function, supporting the concept that Pc colonization results in early, permanent obstructive changes in the lungs of immunosuppressed macaques.

MammaPrint was the first genomic assay in breast cancer to be validated with a prospective randomized trial, the MINDACT trial. The 70 gene MammaPrint assay was developed to determine the risk of distant metastasis in early stage breast cancer through gene expression analysis and was the first FDA cleared genomic assay for breast cancer. The assay identifies primary breast cancers likely to metastasize within the first five years of diagnosis and has clinical utility for helping to determine the expected benefit from adjuvant chemotherapy. The MINDACT Trial was the first trial of a genomic assay in breast cancer to provide prospective, randomized evidence of clinical utility for this important clinical question, identifying a significant proportion of patients who could safely forgo chemotherapy within a cohort of patients with high risk clinical characteristics. Nearly half of all patients (46%) who would have been advised chemotherapy according to clinical guidelines were identified genomically by MammaPrint as being low risk and found to have equivalent rates of freedom from metastasis at 5 years with or without chemotherapy. Based upon the MINDACT trial, the ASCO Biomarker Guidelines now approve the use of MammaPrint to inform decisions regarding chemotherapy for women with clinically high-risk ER+ breast cancer, and as the only approved assay for use in women with 1-3 involved lymph nodes. Recent studies suggest information obtained from the 70-gene assay may also help inform decisions regarding endocrine therapy, as well as chemotherapy, targeted therapy and immunotherapy.Conclusion. The power of gene expression analysis in breast cancer, effectively illustrated with MammaPrint in the MINDACT trial, is now being explored through examination of the full transcriptome in breast cancer.

The current paper discusses the use of genomics in the context of the changing landscape of clinical practice and modern medicine. Medical practice has shifted considerably over the past few decades, from empirical to evidence-based to personalized medicine, and the transition from reliance on observation to measureable parameters. Scientific innovation is required to collect an ever-increasing number and variety of data points and sophisticated analyses capable of distilling vast datasets into meaningful information. The next phase of innovation seeks to personalize disease management, in particular through genomics in oncology. With expanding use of genomics in medicine, and several initiatives collecting genomic data at the population level, education of patients and physicians is critical for data utility. By combining genomic and clinical data, bioinformatics approaches can be applied to developing individualized or targeted therapies. Breast cancer provides an example through which to understand the evolution of genomic data from purescience to clinical utility. From intrinsic subtype classification to development of multigene panels estimating recurrence risk, new studies, such as the FLEX trial, will expand to evaluate the whole transcriptome of tumours. This approach will enable discovery of novel gene signatures and ultimately pave the way toward a personalized approach to breast cancer management.Conclusion. Despite the potential for genomics to personalize treatments, a number of challenges remain to fully integrate these types of large datasets in a manner that provides clinicians and patients with meaningful, actionable information. However, if challenges are addressed, precision medicine has the capacity to transform patient care.

In periodontitis, a common chronic inflammatory condition, gram-negative-rich bacterial biofilms trigger, in susceptible individuals, perpetuating inflammation that results in extensive tissue damage of tooth supporting structures. To delineate immune cell-dependent mechanisms whereby bacterial challenge drives persistent destructive inflammation in periodontitis and other inflammatory diseases, we studied involved tissues ex vivo and investigated host cell responses to the periodontal pathogen Porphyromonas gingivalis, in vitro. Diseased lesions were populated by abundant Th17 cells, linked to infection, chronic inflammation/autoimmunity and tissue pathology. In vitro, P. gingivalis, particularly the more virulent strain W83, stimulated myeloid antigen presenting cells (APC) to drive Th17 polarization. Supernatants from myeloid APC exposed to P. gingivalis were capable of enhancing Th17 but not Th1 polarization. P. gingivalis favored the generation of Th17 responses by stimulating the production of Th17 related cytokines IL-1β, IL-6 and IL-23, but not Th1 related IL-12. By inducing NFκB activation, P. gingivalis promoted IL-1β, IL-6 and IL-12p40 production, but not IRF3 phosphorylation, connected to generation of the IL-12p35 chain, ultimately restricting formation of the intact IL-12 molecule. Promotion of Th17 lineage responses was also aided by P. gingivalis proteases, which appeared to differentially degrade pivotal cytokines. In this regard, IL-12 was largely degraded by P. gingivalis, whereas IL-1β was more resistant to proteolysis. Our data unveil multiple pathways by which P. gingivalis may orchestrate chronic inflammation, providing insights into interventional strategies.

Pneumocystis (Pc) colonization is common among human immunodeficiency virus (HIV)-infected subjects, although the clinical consequences of Pc carriage are not fully understood. We examined the frequency of asymptomatic carriage in healthy and simian immunodeficiency virus (SIV)-infected cynomolgus macaques by use of polymerase chain reaction (PCR) and assessment of changes in the serologic response to a recombinant fragment of the Pc protein kexin (KEX1). Anti-KEX1 antibodies were detected in 95% of healthy monkeys. To create a model of natural transmission of Pc, SIV-infected monkeys were cohoused with macaques coinfected with SIV and Pc. Pc colonization occurred when the CD4(+) T cell count decreased to

Pneumocystis colonization has been associated with severity of chronic obstructive pulmonary disease (COPD). The relationship of Pneumocystis antibody status to COPD severity has not been investigated, but antibody levels might relate to both colonization susceptibility and COPD progression. We investigated anti-Pneumocystis antibody titers and airway obstruction in a cohort of patients with COPD. Undetectable anti-Pneumocystis antibody titer was an independent predictor of more-severe airway obstruction, although use of inhaled corticosteroids is a possible confounder of this effect.

Pulmonary arterial hypertension (PAH) is increased in HIV, but its pathogenesis is not fully understood. Nonhuman primates infected with simian immunodeficiency virus (SIV) or SIV-HIV chimeric virus (SHIV) exhibit histologic changes characteristic of human PAH, but whether hemodynamic changes accompany this pathology is unknown. Repeated measurements of pulmonary artery pressures would permit longitudinal assessments of disease development and provide insights into pathogenesis. We tested the hypothesis that SIV-infected and SHIV-infected macaques develop physiologic manifestations of PAH. We performed right heart catheterizations, echocardiography, and computed tomography (CT) scans in macaques infected with either SIV (ΔB670) or SHIV (89.6P), and compared right heart and pulmonary artery pressures, as well as pulmonary vascular changes on CT scans, with those in uninfected control animals. Right atrial, right ventricular systolic, and pulmonary artery pressures (PAPs) were significantly elevated in 100% of macaques infected with either SIV or SHIV compared with control animals, with no difference in pulmonary capillary wedge pressure. PAPs increased as early as 3 months after SIV infection. Radiographic evidence of pulmonary vascular pruning was also found. Both SIV-infected and SHIV-infected macaques exhibited histologic changes in pulmonary arteries, predominantly consisting of intimal and medial hyperplasia. This report is the first to demonstrate SHIV-infected and SIV-infected macaques develop pulmonary hypertension at a high frequency, with physiologic changes occurring as early as 3 months after infection. These studies establish an important nonhuman primate model of HIV-associated PAH that will be useful in studies of disease pathogenesis and the efficacy of interventions.

Background PAH is a progressive disease of the pulmonary vasculature and right heart, with an increased prevalence in HIV-infected individuals. The causes of HIV-PAH are not known, though evidence suggests immune-mediated pathogenesis. We hypothesize that chronic inflammation contributes to increased pro-inflammatory monocytes and fibrotic alveolar macrophages, which may promote the vascular remodeling characteristic of PAH. We have developed a primate model of simian immunodeficiency virus (SIV)-PAH. This model was used to address the relationship between phenotypic changes in monocyte/macrophage and the development of SIV-PAH. Methods Macaques (n=22) were infected with SIV and serial blood and bronchoalveolar lavage fluid cells were analyzed by flow cytometry. Serial right heart catheterizations were performed to assess mean pulmonary arterial pressure (mPAP). Results Nine macaques showed no change in mPAP over the course of infection, whereas 13 developed PH (mPAP >25mm Hg). Higher frequencies of pro-inflammatory, non-classical monocytes were observed in PH+ animals (p=0.0001), correlating with mPAP (p=0.03). PH- animals had a higher frequency of phagocytic, classical monocytes (p=0.046), negatively correlating with mPAP (p=0.02). PH+ animals had a higher frequency of fibrotic M2a macrophages (p=0.02), correlating with mPAP (p=0.035). PH- animals had a higher frequency of regulatory, M2c macrophages (p=0.0003), negatively correlating with mPAP (p=0.005). Conclusion These results support the hypothesis that HIV-associated PAH is associated with skewing of monocytes and macrophages toward a pro-inflammatory and fibrotic phenotype that contributes to vascular remodeling associated with HIV-PAH.

The lack of animal models of HIV-related pulmonary arterial hypertension (HIV-PAH) severely limits investigation of this serious disease. While histological evidence of HIV-PAH has been demonstrated in macaques infected with simian immunodeficiency virus (SIV) as well as with chimeric simian/human immunodeficiency virus (SHIV) containing HIV-1-derived Nef protein, other primate models have not been studied. The objective was to document and describe the development of pulmonary vascular changes in macaques infected with SIV or with SIV containing HIV-1-derived envelope protein (SHIV-env). Lung tissue was obtained at necropsy from 13 SHIV (89.6P)-env-infected macaques and 10 SIV (ΔB670)-infected macaques. Pulmonary arterial pathology, including arterial hyperplasia and the presence of plexiform lesions, was compared to normal monkey lung. Pulmonary artery hyperplasia was present in 8 of 13 (62%) SHIV-env-infected macaques and 4/10 (36%) SIV-infected macaques. The most common histopathological lesions were intimal and medial hyperplasia of medium and large pulmonary arteries. Hyperplastic lesions were predominantly due to smooth muscle cell hyperplasia. This is the first report of pulmonary vascular lesions in SHIV-env-infected macaques and confirms prior reports of pulmonary vasculopathy in SIV-infected macaques. The finding of pulmonary arteriopathy in monkeys infected with SHIV not containing HIV-nef suggests that other factors might also be important in the development of HIV-PAH. This SHIV-env model provides a new means to investigate HIV-PAH.

Pulmonary colonization by the opportunistic pathogen Pneumocystis jiroveci is common in HIV + subjects and has been associated with development of chronic obstructive pulmonary disease (COPD). Host and environmental factors associated with colonization susceptibility are undefined. Using a simian-human immunodeficiency virus (SHIV) model of HIV infection, the immunologic parameters associated with natural Pneumocystis jiroveci transmission were evaluated. SHIV-infected macaques were exposed to P. jiroveci by cohousing with immunosuppressed, P. jiroveci -colonized macaques in two independent experiments. Serial plasma and bronchoalveolar lavage (BAL) fluid samples were examined for changes in antibody titers to recombinant Pneumocystis -kexin protein (KEX1) and evidence of Pneumocystis colonization by nested PCR of BAL fluid. In experiment 1, 10 of 14 monkeys became Pneumocystis colonized (Pc + ) by 8 weeks post-SHIV infection, while 4 animals remained Pneumocystis colonization negative (Pc − ) throughout the study. In experiment 2, 11 of 17 animals became Pneumocystis colonized by 16 weeks post-SHIV infection, while 6 monkeys remained Pc − . Baseline plasma KEX1-IgG titers were significantly higher in monkeys that remained Pc − , compared to Pc + monkeys, in experiments 1 ( P = 0.013) and 2 ( P = 0.022). Pc − monkeys had greater percentages of Pneumocystis -specific memory B cells after SHIV infection compared to Pc + monkeys ( P = 0.037). After SHIV infection, Pc + monkeys developed progressive obstructive pulmonary disease, whereas Pc − monkeys maintained normal lung function throughout the study. These results demonstrate a correlation between the KEX1 humoral response and the prevention of Pneumocystis colonization and obstructive lung disease in the SHIV model. In addition, these results indicate that an effective Pneumocystis -specific memory B-cell response is maintained despite progressive loss of CD4 + T cells during SHIV infection.

Human immunodeficiency virus (HIV)-infected patients are at increased risk for development of pulmonary complications, including chronic obstructive pulmonary disease (COPD). Inflammation associated with subclinical infection has been postulated to promote COPD. Persistence of Pneumocystis is associated with HIV infection and COPD, although a causal relationship has not been established. We used a simian/human immunodeficiency virus model of HIV infection to study pulmonary effects of Pneumocystis colonization. Simian/human immunodeficiency virus-infected/Pneumocystis-colonized monkeys developed progressive obstructive pulmonary disease characterized by increased emphysematous tissue and bronchial-associated lymphoid tissue. Increased levels of T helper type 2 cytokines and proinflammatory mediators in bronchoalveolar lavage fluid coincided with Pneumocystis colonization and a decline in pulmonary function. These results support the concept that an infectious agent contributes to the development of HIV-associated lung disease and suggest that Pneumocystis colonization may be a risk factor for the development of HIV-associated COPD. Furthermore, this model allows examination of early host responses important to disease progression, thus identifying potential therapeutic targets for COPD.

Background Longitudinal studies of the lung microbiome are challenging due to the invasive nature of sample collection. In addition, studies of the lung microbiome in human disease are usually performed after disease onset, limiting the ability to determine early events in the lung. We used a non-human primate model to assess lung microbiome alterations over time in response to an HIV-like immunosuppression and determined impact of the lung microbiome on development of obstructive lung disease. Cynomolgous macaques were infected with the SIV-HIV chimeric virus SHIV89.6P. Bronchoalveolar lavage fluid samples were collected pre-infection and every 4 weeks for 53 weeks post-infection. The microbiota was characterized at each time point by 16S ribosomal RNA (rRNA) sequencing. Results We observed individual variation in the composition of the lung microbiota with a proportion of the macaques having Tropheryma whipplei as the dominant organism in their lungs. Bacterial communities varied over time both within and between animals, but there did not appear to be a systematic alteration due to SHIV infection. Development of obstructive lung disease in the SHIV-infected animals was characterized by a relative increase in abundance of oral anaerobes. Network analysis further identified a difference in community composition that accompanied the development of obstructive disease with negative correlations between members of the obstructed and non-obstructed groups. This emphasizes how species shifts can impact multiple other species, potentially resulting in disease. Conclusions This study is the first to investigate the dynamics of the lung microbiota over time and in response to immunosuppression in a non-human primate model. The persistence of oral bacteria in the lung and their association with obstruction suggest a potential role in pathogenesis. The lung microbiome in the non-human primate is a valuable tool for examining the impact of the lung microbiome in human health and disease. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0183-0) contains supplementary material, which is available to authorized users.