Kalari, Krishna R.

Background: Age is the most common risk factor for Alzheimer’s disease (AD), a neurodegenerative disorder characterized by the hallmarks of toxic amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles. Moreover, sub-physiological brain insulin levels have emerged as a pathological manifestation of AD. Objective: Identify age-related changes in the plasma disposition and blood-brain barrier (BBB) trafficking of Aβ peptides and insulin in mice. Methods: Upon systemic injection of 125I-Aβ40, 125I-Aβ42, or 125I-insulin, the plasma pharmacokinetics and brain influx were assessed in wild-type (WT) or AD transgenic (APP/PS1) mice at various ages. Additionally, publicly available single-cell RNA-Seq data [GSE129788] was employed to investigate pathways regulating BBB transport in WT mice at different ages. Results: The brain influx of 125I-Aβ40, estimated as the permeability-surface area product, decreased with age, accompanied by an increase in plasma AUC. In contrast, the brain influx of 125I-Aβ42 increased with age, accompanied by a decrease in plasma AUC. The age-dependent changes observed in WT mice were accelerated in APP/PS1 mice. As seen with 125I-Aβ40, the brain influx of 125I-insulin decreased with age in WT mice, accompanied by an increase in plasma AUC. This finding was further supported by dynamic single-photon emission computed tomography (SPECT/CT) imaging studies. RAGE and PI3K/AKT signaling pathways at the BBB, which are implicated in Aβ and insulin transcytosis, respectively, were upregulated with age in WT mice, indicating BBB insulin resistance. Conclusion: Aging differentially affects the plasma pharmacokinetics and brain influx of Aβ isoforms and insulin in a manner that could potentially augment AD risk.

Objective To test the ability of machine learning (ML) approaches with clinical and genomic biomarkers to predict methotrexate treatment response in patients with early rheumatoid arthritis (RA). Methods Demographic, clinical, and genomic data from 643 patients of European ancestry with early RA (mean age 54 years; 70% female) subdivided into a training (n = 336) and validation cohort (n = 307) were used. The genomic data comprised 160 single‐nucleotide polymorphisms (SNPs) previously associated with RA or methotrexate metabolism. Response to methotrexate monotherapy was defined as good or moderate by the European Alliance of Associations for Rheumatology (EULAR) response criteria at the 3‐month follow‐up. Supervised ML methods were trained with 5 repeats and 10‐fold cross‐validation using the training cohort. Prediction performance was validated in the independent validation cohort. Results Supervised ML methods combining age, sex, smoking, rheumatoid factor, baseline Disease Activity Score in 28 joints (DAS28) scores and 160 SNPs predicted EULAR response at 3 months with the area under the receiver operating curve of 0.84 ( P = 0.05) in the training cohort and achieved a prediction accuracy of 76% ( P = 0.05) in the validation cohort (sensitivity 72%, specificity 77%). Intergenic SNPs rs12446816, rs13385025, rs113798271, and ATIC (rs2372536) had variable importance above 60.0 and along with baseline DAS28 scores were among the top predictors of methotrexate response. Conclusion Pharmacogenomic biomarkers combined with baseline DAS28 scores can be useful in predicting response to methotrexate in patients with early RA. Applying ML to predict treatment response holds promise for guiding effective RA treatment choices, including timely escalation of RA therapies.

Abstract Our previous matched case–control study of postmenopausal women with resected early-stage breast cancer revealed that only anastrozole, but not exemestane or letrozole, showed a significant association between the 6-month estrogen concentrations and risk of breast cancer. Anastrozole, but not exemestane or letrozole, is a ligand for estrogen receptor α. The mechanisms of endocrine resistance are heterogenous and with the new mechanism of anastrozole, we have found that treatment of anastrozole maintains fatty acid synthase (FASN) protein level by limiting the ubiquitin-mediated FASN degradation, leading to increased breast cancer cell growth. Mechanistically, anastrozole decreases the guided entry of tail-anchored proteins factor 4 (GET4) expression, resulting in decreased BCL2-associated athanogene cochaperone 6 (BAG6) complex activity, which in turn, prevents RNF126-mediated degradation of FASN. Increased FASN protein level can induce a negative feedback loop mediated by the MAPK pathway. High levels of FASN are associated with poor outcome only in patients with anastrozole-treated breast cancer, but not in patients treated with exemestane or letrozole. Repressing FASN causes regression of breast cancer cell growth. The anastrozole-FASN signaling pathway is eminently targetable in endocrine-resistant breast cancer.

Abstract The role of maternal microbiome transmitted at birth in cancer control is poorly understood. We have developed the first germfree B6 mouse model of breast cancer to investigate the role of the maternal microbiome in controlling oncogenic/metastatic frequencies of pro-oncogenic mammary cells. In this model, a DNA barcoded, primitive normal mouse mammary epithelial cell encoding MMTV-PyMT oncogene, was transplanted in large numbers into conventional or germfree B6 mice. Next-gen sequencing analysis of the DNA barcodes in tissues enabled us to clonally track millions of cells and measure the frequency and growth dynamics of clones at the primary site and their systemic distribution in circulation and all vital organs, generating an unprecedented high-definition map of cancer progression. Our results show that in conventional B6 mice with maternal microbial transmission at-birth, a small fraction (~0.01%) of transplanted cells transform de novo and produce slow growing, late-onset benign tumors (mouse median survival of > 1 year). In contrast, in germfree B6 mice, a >10-fold higher frequency of cells transform de novo and generate early-onset, highly aggressive metastatic clones, and are frequently associated with features leading to early euthanasia or endpoint (i.e. sudden death, intracardiac metastasis, paralysis, swollen abdomen, early multiorgan aggressive metastasis) (median survival of ~4 months; p<0.005 vs conventional mice). However, postnatal intervention with healthy fecal implants reconstituted gut microbiota in germfree B6 mice and significantly restored protective effects against cancer progression and sudden death, as well as improved quality/duration of life (median survival of >1 year; p<0.005 vs germfree mice; p=ns vs conventional mice). We found maternal microbial transmission fully protected against systemic metastasis. GC-MS analysis identified significantly distinct primary tumor metabolome associated with conventional (non-metastatic) mice vs germfree (metastatic) mice. Metagenomic analysis of fecal samples from a subset of tumor bearing conventional (non-metastatic) mice vs fecal implanted (late-onset, less aggressive metastatic) germfree mice identified significant association between metastasis and altered microbial communities. These findings using novel germfree mouse model of metastasis and cellular DNA barcoding technology provide first direct evidence that diverse microbial communities transmitted at birth play a critical role in protecting the host from clonal de novo mammary cell transformation and progression to systemic disease and thereby improving quality and duration of life. Citation Format: Nagarajan Kannan, Syed Mohammed Musheer Aalam, Xiaojia Tang, Krishna Kalari, Purna Kashyap, Jun Chen, Stephen Johnson, Anguraj Sadanandam, Mark Sherman. Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB226.

Thiopurine drugs such as 6‐mercaptopurine (6‐MP) and 6‐thioguanine (6‐TG) are effective immunosuppressants and antineoplastic agents. We used a genome‐wide approach to identify genes that might contribute to individual variation in thiopurine response. Basal gene expression profiles, genome‐wide SNP data, and 6‐MP and 6‐TG Cell growth inhibition (GI50) values were generated for 200 Coriell Institute “Human Variation Panel” lymphoblastoid cell lines. Gene expression and genome‐wide SNPs were correlated with GI50 values to identify candidate genes that might contribute to thiopurine response. We identified 104 expression probes and 161 SNPs, as well as 315 gene probes and 579 SNPs that correlated significantly with GI50 values for 6‐MP and 6‐TG, respectively (P<10 −5 and 10 −6 , respectively). Three genes, CD83 , TXNDC13 and SNAP23 , contained SNPs that contributed significantly to both 6‐MP and 6‐TG cytotoxicity and were also significantly associated with expression levels (P<10 −4 ), suggesting a “three‐way model” involving genotype, gene expression and cytotoxicity. GBP5 , a gene with an expression level that correlated negatively with 6‐MP and 6‐TG GI50 values, sensitized leukemia cells when overexpressed. Functional studies of the remainder of the candidate genes are being performed. These results may enhance our ability to predict thiopurine response phenotypes. NIH GM28157.

Objectives Betaine‐homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT in the human liver is responsible for up to 25% of homocysteine metabolism. Knowledge of the relationship between genetic polymorphisms and BHMT phenotypes could enhance our understanding of the role of this reaction in drug metabolism. Methods Levels of BHMT enzyme activity and immunoreactive protein were measured in 268 human liver biopsies. Twelve tag BHMT SNPs from our own gene resequencing and HapMap data were used to genotype DNA from these samples. Genotype‐phenotype association analysis was then performed. BHMT enzyme activity averaged 0.077 ± 0.028 units/μg, with a 12 fold variation. Results BHMT protein levels averaged 6.9 ± 3.3 units/μg, with a 50‐fold variation. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28kb upstream, in the 5′‐UTR and in intron 1 of BHMT , respectively, were significantly correlated with both phenotypes (p<10 −8 for enzyme activity and p<10 −5 for protein level). rs41272270 was associated with altered protein binding during electrophoresis mobility shift assay. Conclusions Three tag SNPs were significantly associated with human hepatic BHMT enzyme activity and immunoreactive protein. These observations may help us understand the regulation of BHMT in the human liver and its role in drug metabolism. Supported by NIH U01 GM61388, R01 GM28157, R01 CA132780.

Abstract Purpose: Programmed death ligand 1 [PD-(L)1]-targeted therapies have shown modest survival benefit in triple-negative breast cancer (TNBC). PD-L1+ microenvironments in TNBC are not well characterized and may inform combinatorial immune therapies. Herein, we characterized clinicopathologic features, RNA-based immune signatures, and spatially defined protein-based tumor–immune microenvironments (TIME) in early-stage PD-L1+ and PD-L1− TNBC. Experimental Design: From a large cohort of chemotherapy-naïve TNBC, clinicopathologic features, deconvoluted RNA immune signatures, and intraepithelial and stromal TIME (Nanostring GeoMX) were identified in subsets of PD-L1+ and PD-L1− TNBC, as defined by FDA-approved PD-L1 companion assays. Results: 228 of 499 (46%) TNBC were PD-L1+ (SP142: ≥1% immune cells-positive). Using PD-L1 22C3, 46% had combined positive score (CPS) ≥ 1 and 16% had CPS ≥10. PD-L1+ TNBC were higher grade with higher tumor-infiltrating lymphocytes (TIL; P &lt; 0.05). PD-L1 was not associated with improved survival following adjustment for TILs and other variables. RNA profiles of PD-L1+ TNBC had increased dendritic cell, macrophage, and T/B cell subset features; and decreased myeloid-derived suppressor cells. PD-L1+ stromal and intraepithelial TIMEs were highly enriched in IDO-1, HLA-DR, CD40, and CD163 compared with PD-L1-TIME, with spatially specific alterations in CTLA-4, Stimulator of Interferon Genes (STING), and fibronectin. Macrophage- and antigen presentation–related proteins correlated most strongly with PD-L1 protein. Conclusions: In this early-stage TNBC cohort, nearly 50% were PD-L1+ (SP142 companion assay) while 16% were PD-L1+ with the 22C3 companion assay. PD-L1+ TNBC had specific myeloid-derived and lymphoid features. Spatially defined PD-L1+ TIME were enriched in several clinically actionable immune proteins. These data may inform future studies on combinatorial immunotherapies for patients with PD-L1+ TNBC. See related commentary by Symmans, p. 5446

Abstract Purpose: Patient-derived xenografts (PDX) are a research tool for studying cancer biology and drug response phenotypes. While engraftment rates are higher for tumors with more aggressive characteristics, it is uncertain whether engraftment is prognostic for cancer recurrence. Patients and Methods: In a prospective study of patients with breast cancer treated with neoadjuvant chemotherapy (NAC) with taxane ± trastuzumab followed by anthracycline-based chemotherapy, we report the association between breast cancer events and PDX engraftment using tumors derived from treatment naïve (pre-NAC biopsies from 113 patients) and treatment resistant (post-NAC at surgery from 34 patients). Gray test was used to assess whether the cumulative incidence of a breast cancer event differs with respect to either pre-NAC PDX engraftment or post-NAC PDX engraftment. Results: With a median follow-up of 5.7 years, the cumulative incidence of breast cancer relapse did not differ significantly according to pre-NAC PDX engraftment (5-year rate: 13.6% vs. 13.4%; P = 0.89). However, the incidence of a breast event was greater for patients with post-NAC PDX engraftment (5-year rate: 50.0% vs. 19.6%), but this did not achieve significance (P = 0.11). Conclusions: In treatment-naïve breast cancer receiving standard NAC, PDX engraftment was not prognostic for breast cancer recurrence. Further study is needed to establish whether PDX engraftment in the treatment-resistant setting is prognostic for cancer recurrence.

548 Background: sTILs are a well-established prognostic and predictive biomarker in patients with operable TNBC receiving pre or postoperative systemic therapy. We1 and others2,3 have also shown that sTILs are prognostic in patients who did not receive adjuvant chemotherapy. Here, we detail the outcomes of systemically untreated patients with N0 TNBC according to sTIL score. We focused on the N0 subset as a group of patients who may be candidates for future prospective therapy de-escalation trials. Methods: From a clinically annotated cohort of 605 patients with centrally confirmed TNBC (ER/PR < 1% and HER2 negative) with long-term outcomes data, we identified 182 patients treated with locoregional therapy only (breast surgery +/- radiation therapy and no chemotherapy). The clinicopathological characteristics of this cohort have previously been published1. In this analysis, we report the 5- and 10-year invasive disease-free survival (iDFS) and overall survival (OS) rates of patients with N0 TNBC according to sTIL levels. IDFS and OS were defined as per the STEEP classification and estimated using the Kaplan–Meier method. Comparisons of the survival distributions between groups were assessed by the log-rank test. sTILs were assessed as a continuous parameter according to the International TIL Working Group guidelines. For comparisons of outcomes between groups, tumors were classified as lymphocyte-predominant TNBC (defined as containing ≥50% sTILs) vs non-lymphocyte-predominant ( < 50% sTILs). Results: Of 182 systemically untreated patients, 149 (82%) were N0 and most (78%) were post-menopausal. T stage distribution was T1: 68%, T2: 28%, T3/4: 4%. Among N0 patients, 31 (21%) had lymphocyte-predominant TNBC, and in this group the 5-year iDFS and OS were 89% (95% CI 76-100) and 96% (95% CI 89-100), while the 10-year iDFS and OS were 89% (95% CI 76-100) and 87% (95% CI 73-100), respectively. In contrast, outcomes for patients with non-lymphocyte predominant TNBC were significantly worse. For this group, 5-year iDFS and OS were 62% (95% CI 53-73) and 78% (95% CI 71-86) while the 10-year iDFS and OS were 45% (95% CI 36-58) and 66% (95% CI 68-76), respectively ( log-rank p = 0.02 for iDFS and log-rank p = 0.03 for OS). Conclusions: sTIL quantification identifies a subset of patients with early-stage N0 TNBC with an exceedingly good prognosis, even in the absence of adjuvant chemotherapy. These data provide support for the evaluation of sTILs as part of prospective investigation of systemic therapy de-escalation strategies in N0 TNBC. References:1Leon-Ferre et al, Breast Cancer Res Treat (2018) 167:89-99 2Park et al, Ann Oncol (2019) 12:1941-1949 3De Jong et al, ESMO 2020

Aromatase inhibitors (AIs) are the treatment of choice for hormone receptor–positive early breast cancer in postmenopausal women. None of the third‐generation AIs are superior to the others in terms of efficacy. We attempted to identify genetic factors that could differentiate between the effectiveness of adjuvant anastrozole and exemestane by examining single‐nucleotide polymorphism (SNP)‐treatment interaction in 4,465 patients. A group of SNPs were found to be differentially associated between anastrozole and exemestane regarding outcomes. However, they showed no association with outcome in the combined analysis. We followed up common SNPs near LY75 and GPR160 that could differentiate anastrozole from exemestane efficacy. LY75 and GPR160 participate in epithelial‐to‐mesenchymal transition and growth pathways, in both cases with SNP‐dependent variation in regulation. Collectively, these studies identified SNPs that differentiate the efficacy of anastrozole and exemestane and they suggest additional genetic biomarkers for possible use in selecting an AI for a given patient.