An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics.

For a decade, The Cancer Genome Atlas (TCGA) program collected clinicopathologic annotation data along with multi-platform molecular profiles of more than 11,000 human tumors across 33 different cancer types. TCGA clinical data contain key features representing the democratized nature of the data collection process. To ensure proper use of this large clinical dataset associated with genomic features, we developed a standardized dataset named the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR), which includes four major clinical outcome endpoints. In addition to detailing major challenges and statistical limitations encountered during the effort of integrating the acquired clinical data, we present a summary that includes endpoint usage recommendations for each cancer type. These TCGA-CDR findings appear to be consistent with cancer genomics studies independent of the TCGA effort and provide opportunities for investigating cancer biology using clinical correlates at an unprecedented scale.

Cell-of-Origin Patterns Dominate the Molecular Classification of 10,000 Tumors from 33 Types of Cancer.

We conducted comprehensive integrative molecular analyses of the complete set of tumors in The Cancer Genome Atlas (TCGA), consisting of approximately 10,000 specimens and representing 33 types of cancer. We performed molecular clustering using data on chromosome-arm-level aneuploidy, DNA hypermethylation, mRNA, and miRNA expression levels and reverse-phase protein arrays, of which all, except for aneuploidy, revealed clustering primarily organized by histology, tissue type, or anatomic origin. The influence of cell type was evident in DNA-methylation-based clustering, even after excluding sites with known preexisting tissue-type-specific methylation. Integrative clustering further emphasized the dominant role of cell-of-origin patterns. Molecular similarities among histologically or anatomically related cancer types provide a basis for focused pan-cancer analyses, such as pan-gastrointestinal, pan-gynecological, pan-kidney, and pan-squamous cancers, and those related by stemness features, which in turn may inform strategies for future therapeutic development.

Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin.

Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head and neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multiplatform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All data sets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies.

Quantifying hormone receptor status in lobular breast cancer in an institutional series: the relationship between estrogen and progesterone receptor status and outcomes.

PURPOSE: Recent guidelines defined a new reporting category of ER-low-positive breast cancer based on immunohistochemistry (IHC). While low positivity of either hormone receptor is uncommon in invasive lobular carcinoma (ILC), we sought to investigate whether relatively low hormone receptor positivity was associated with tumor characteristics and patient outcomes in a single institutional cohort. METHODS: We searched an institutional database for cases of stage I-III ILC with available IHC reports. Based on prior published categories in ILC, ER was classified as low, medium, or high as defined by ER staining of 10-69%, 70-89%, and ≥ 90% respectively. PR low and high tumors were defined by < 20%, or ≥ 20% staining respectively. We used chi-squared tests, t-tests, and Cox proportional hazards models to evaluate associations between ER/PR categories and tumor characteristics or disease-free survival (DFS). RESULTS: The cohort consisted of 707 ILC cases, with 11% of cases categorized as ER low, 15.1% as medium, and 73.8% as high. The majority (67.6%) were PR high. Patients with ER low/medium expression were significantly younger, and more likely to also have PR low and/or HER2 positive tumors compared to those that were ER high. In a Cox proportional hazards model adjusting for age, stage, grade, pleomorphic histology, and treatment, ER category was not prognostic for DFS, but PR negative and PR low status each had significantly worse DFS compared to PR high status (HR 3.5, 95% CI 1.8-6.7, p < 0.001; and HR 2.0, 95% CI 1.1-3.5, p = 0.015, respectively). CONCLUSION: These findings highlight the relevance of quantifying ER and PR within ILC.

Clinical and molecular characteristics of estrogen receptor-positive ultralow risk breast cancer tumors identified by the 70-gene signature.

The metastatic potential of estrogen receptor (ER)-positive breast cancers is heterogeneous and distant recurrences occur months to decades after primary diagnosis. We have previously shown that patients with tumors classified as ultralow risk by the 70-gene signature have a minimal long-term risk of fatal breast cancer. Here, we evaluate the previously unexplored underlying clinical and molecular characteristics of ultralow risk tumors in 538 ER-positive patients from the Stockholm tamoxifen randomized trial (STO-3). Out of the 98 ultralow risk tumors, 89% were luminal A molecular subtype, whereas 26% of luminal A tumors were of ultralow risk. Compared to other ER-positive tumors, ultralow risk tumors were significantly (Fisher's test, P < .05) more likely to be of smaller tumor size, lower grade, progesterone receptor (PR)-positive, human epidermal growth factor 2 (HER2)-negative and have low Ki-67 levels (proliferation-marker). Moreover, ultralow risk tumors showed significantly lower expression scores of multi-gene modules associated with the AKT/mTOR-pathway, proliferation (AURKA), HER2/ERBB2-signaling, IGF1-pathway, PTEN-loss and immune response (IMMUNE1 and IMMUNE2) and higher expression scores of the PIK3CA-mutation-associated module. Furthermore, 706 genes were significantly (FDR < 0.001) differentially expressed in ultralow risk tumors, including lower expression of genes involved in immune response, PI3K/Akt/mTOR-pathway, histones, cell cycle, DNA repair, apoptosis and higher expression of genes coding for epithelial-to-mesenchymal transition and homeobox proteins, among others. In conclusion, ultralow risk tumors, associated with minimal long-term risk of fatal disease, differ from other ER-positive tumors, including luminal A molecular subtype tumors. Identification of these characteristics is important to improve our prediction of nonfatal vs fatal breast cancer.

Reduced cardiovascular risks in women with endometriosis or polycystic ovary syndrome carrying a common functional IGF1R variant.

STUDY QUESTION: Is the increased future cardiovascular risk seen in women with endometriosis or polycystic ovary syndrome (PCOS) mitigated by functional insulin-like growth factor-1 receptor (IGF1R) single-nucleotide polymorphism (SNP) rs2016347 as previously shown in women with hypertensive disorders of pregnancy? SUMMARY ANSWER: This cohort study found that women with endometriosis or PCOS who carry a T allele of IGF1R SNP rs2016347 had a reduced future risk of developing cardiovascular disease (CVD) and associated risk factors, with risk reduction dependent on cohort era. WHAT IS KNOWN ALREADY: Women with endometriosis or PCOS have been shown to have an increased future risk of CVD and associated risk factors with limited predictive ability. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study took place in the Nurses' Health Study 2 (NHS2), which enrolled 116 430 participants in 1989 who were followed through 2015. The study population was analyzed in its entirety, and subdivided into entry (pre-1989) and after entry (post-1989) exposure cohorts. All NHS2 participants were eligible for inclusion in the study, 9599 (8.2%) were excluded for missing covariates. PARTICIPANTS/MATERIALS, SETTING, METHODS: The NHS2 enrolled female registered nurses from 14 different states who ranged in age from 25 to 42 years at study entry. Data were collected from entry and biennial questionnaires, and analysis conducted from November 2020 to June 2021. Cox proportional hazard models were used to assess risk of CVD, hypertension (HTN), hypercholesterolemia (HC) and type 2 diabetes, both with and without genotyping for rs2016347. MAIN RESULTS AND THE ROLE OF CHANCE: While women without endometriosis or PCOS, as a whole, demonstrated no impact of genotype on risk in either cohort, women with endometriosis carrying a T allele had a lower risk of CVD (hazard ratio (HR), 0.48; 95% CI, 0.27-0.86, P = 0.02) and HTN (HR, 0.80; 95% CI, 0.66-0.97, P = 0.03) in the pre-1989 cohort, while those in the post-1989 cohort had a decrease in risk for HC (HR, 0.76; 95% CI, 0.62-0.94, P = 0.01). Women with PCOS in the post-1989 cohort showed a significant protective impact of the T allele on HTN (HR, 0.44; 95% CI, 0.27-0.73, P = 0.002) and HC (HR, 0.62; 95% CI, 0.40-0.95, P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Data on specific endometriosis lesion locations or disease stage, as well as on PCOS phenotypes were lacking. In addition, data on systemic medical treatments beyond the use of oral contraceptives were missing, and these treatments may have confounded the results. WIDER IMPLICATIONS OF THE FINDINGS: These findings implicate systemic dysregulation of the insulin-like growth factor-1 axis in the development of HTN, HC and clinical CVD in endometriosis and PCOS, suggesting a common underlying pathogenetic mechanism. STUDY FUNDING/COMPETING INTEREST(S): The NHS2 infrastructure for questionnaire data collection was supported by National Institute of Health (NIH) grant U01CA176726. This work was also supported in part by NIH and National Cancer Institute grant U24CA210990; as well, research effort and publication costs were supported by the Elizabeth MA Stevens donor funds provided to the Buck Institute for Research on Aging. The authors declare they have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties.

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood-brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

A risk-associated Active transcriptome phenotype expressed by histologically normal human breast tissue and linked to a pro-tumorigenic adipocyte population.

BACKGROUND: Previous studies have identified and validated a risk-associated Active transcriptome phenotype commonly expressed in the cancer-adjacent and histologically normal epithelium, stroma, and adipose containing peritumor microenvironment of clinically established invasive breast cancers, conferring a 2.5- to 3-fold later risk of dying from recurrent breast cancer. Expression of this Active transcriptome phenotype has not yet been evaluated in normal breast tissue samples unassociated with any benign or malignant lesions; however, it has been associated with increased peritumor adipocyte composition. METHODS: Detailed histologic and transcriptomic (RNAseq) analyses were performed on normal breast biopsy samples from 151 healthy, parous, non-obese (mean BMI = 29.60 ± 7.92) women, ages 27-66 who donated core breast biopsy samples to the Komen Tissue Bank, and whose average breast cancer risk estimate (Gail score) at the time of biopsy (1.27 ± 1.34) would not qualify them for endocrine prevention therapy. RESULTS: Full genome RNA sequencing (RNAseq) identified 52% (78/151) of these normal breast samples as expressing the Active breast phenotype. While Active signature genes were found to be most variably expressed in mammary adipocytes, donors with the Active phenotype had no difference in BMI but significantly higher Gail scores (1.46 vs. 1.18; p = 0.007). Active breast samples possessed 1.6-fold more (~ 80%) adipocyte nuclei, larger cross-sectional adipocyte areas (p < 0.01), and 0.5-fold fewer stromal and epithelial cell nuclei (p < 1e-6). Infrequent low-level expression of cancer gene hotspot mutations was detected but not enriched in the Active breast samples. Active samples were enriched in gene sets associated with adipogenesis and fat metabolism (FDR q ≤ 10%), higher signature scores for cAMP-dependent lipolysis known to drive breast cancer progression, white adipose tissue browning (Wilcoxon p < 0.01), and genes associated with adipocyte activation (leptin, adiponectin) and remodeling (CAV1, BNIP3), adipokine growth factors (IGF-1, FGF2), and pro-inflammatory fat signaling (IKBKG, CCL13). CONCLUSIONS: The risk-associated Active transcriptome phenotype first identified in cancer-adjacent breast tissues also occurs commonly in healthy women without breast disease who do not qualify for breast cancer chemoprevention, and independently of breast expressed cancer-associated mutations. The risk-associated Active phenotype appears driven by a pro-tumorigenic adipocyte microenvironment that can predate breast cancer development.

Integrated genomic characterization of endometrial carcinoma.

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ∼25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours.

Tamoxifen therapy benefit for patients with 70-gene signature high and low risk.

BACKGROUND: Breast cancer molecular prognostic tools that predict recurrence risk have mainly been established on endocrine-treated patients and thus are not optimal for the evaluation of benefit from endocrine therapy. The Stockholm tamoxifen (STO-3) trial which randomized postmenopausal node-negative patients to 2-year tamoxifen (followed by an optional randomization for an additional 3-year tamoxifen vs nil), versus no adjuvant treatment, provides a unique opportunity to evaluate long-term 20-year benefit of endocrine therapy within prognostic risk classes of the 70-gene prognosis signature that was developed on adjuvantly untreated patients. METHODS: We assessed by Kaplan-Meier analysis 20-year breast cancer-specific survival (BCSS) and 10-year distant metastasis-free survival (DMFS) for 538 estrogen receptor (ER)-positive, STO-3 trial patients with retrospectively ascertained 70-gene prognosis classification. Multivariable analysis of long-term (20 years) BCSS by STO-3 trial arm in the 70-gene high-risk and low-risk subgroups was performed using Cox proportional hazard modeling adjusting for classical patient and tumor characteristics. RESULTS: Tamoxifen-treated, 70-gene low- and high-risk patients had 20-year BCSS of 90 and 83%, as compared to 80 and 65% for untreated patients, respectively (log-rank p < 0.0001). Notably, there is equivalent tamoxifen benefit in both high (HR 0.42 (0.21-0.86), p = 0.018) and low (HR 0.46 (0.25-0.85), p = 0.013) 70-gene risk categories even after adjusting for clinico-pathological factors for BCSS. Limited tamoxifen exposure as given in the STO-3 trial provides persistent benefit for 10-15 years after diagnosis in a time-varying analysis. 10-year DMFS was 93 and 85% for low- and high-risk tamoxifen-treated, versus 83 and 70% for low- and high-risk untreated patients, respectively (log-rank p < 0.0001). CONCLUSIONS: Patients with ER-positive breast cancer, regardless of high or low 70-gene risk classification, receive significant survival benefit lasting over 10 years from adjuvant tamoxifen therapy, even when given for a relatively short duration.

Functional IGF1R variant predicts breast cancer risk in women with preeclampsia in California Teachers Study.

PURPOSE: Hypertension in pregnancy has been associated with decreased future risk of breast cancer in many but not all studies. In the Marin Women's Study, pregnancy-induced hypertension was shown to interact with the T allele of a functional IGF1R gene variant, rs2016347, to result in lower breast density, as well as decreased breast cancer risk. Our objective was to explore these findings in a larger sample of women from the California Teachers Study (CTS). METHODS: The CTS cohort consists of over 130,000 female educators. DNA was available from a nested case-control study, which included 2,030 non-Hispanic white women who developed breast cancer and 1,552 controls. The current study included all participants from the case-control group with a self-reported history of preeclampsia (80 cases/57 controls). RESULTS: Comparing TT to GG genotypes revealed adjusted odds ratios of 0.38 (CI 0.13, 1.14) for all invasive breast cancers, 0.26 (CI 0.07, 0.89) for hormone receptor-positive (HR+) breast cancers, 0.15 (CI 0.04, 0.56) for those with age at first birth (AFB) < 30, and 0.10 (CI 0.02, 0.49) for those with AFB < 30 and HR+ breast cancers. Trend analysis yielded p values of 0.09, 0.03, 0.005, and 0.004 respectively, suggesting a biological effect for each T allele. CONCLUSION: Study findings indicate that the T allele of IGF1R variant rs2016347 is associated with a significant reduction in breast cancer risk in women with a history of preeclampsia, most marked for HR+ breast cancer and in women with AFB < 30.

Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma.

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.

FOXM1 cistrome predicts breast cancer metastatic outcome better than FOXM1 expression levels or tumor proliferation index.

FOXM1 is a key transcription factor regulating cell cycle progression, DNA damage response, and a host of other hallmark cancer features, but the role of the FOXM1 cistrome in driving estrogen receptor-positive (ER+) versus estrogen receptor-negative (ER-) breast cancer clinical outcomes remains undefined. Chromatin immunoprecipitation sequencing (ChIP-Seq) coupled with RNA sequencing (RNA-Seq) analyses was used to identify FOXM1 target genes in breast cancer cells (MCF-7) where FOXM1 expression was either induced by cell proliferation or repressed by p53 upregulation. The prognostic performance of these FOXM1 target genes was assessed relative to FOXM transcript levels and a 61-gene proliferation score (PS) for their ability to dichotomize a pooled cohort of 683 adjuvant chemotherapy-naïve, node-negative breast cancer cases (447 ER+, 236 ER-). Differences in distant metastasis-free survival (DMFS) between the dichotomized expression groups were determined by Cox proportional hazard modeling. Proliferation-associated FOXM1 upregulation induced a set of 145 differentially bound and expressed genes (direct targets), and these demonstrated minimal overlap with differentially bound and expressed genes following FOXM1 repression by p53 upregulation. This proliferation-associated FOXM1 cistrome was not only better at significantly predicting metastatic outcome of ER+ breast cancers (HR: 2.8 (2.0-3.8), p = 8.13E-10), but was the only parameter trending toward significance in predicting ER- metastatic outcome (HR: 1.6 (0.9-2.9), p = 0.087). Our findings demonstrate that FOXM1 target genes are highly dependent on the cellular context in which FOXM1 expression is modulated, and a newly identified proliferation-associated FOXM1 cistromic signature best predicts breast cancer metastatic outcome.

mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms.

Resistance of breast cancers to targeted hormone receptor (HR) or human epidermal growth factor receptor 2 (HER2) inhibitors often occurs through dysregulation of the phosphoinositide 3-kinase, protein kinase B/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Presently, no targeted therapies exist for breast cancers lacking HR and HER2 overexpression, many of which also exhibit PI3K/AKT/mTOR hyper-activation. Resistance of breast cancers to current therapeutics also results, in part, from aberrant epigenetic modifications including protein acetylation regulated by histone deacetylases (HDACs). We show that the investigational drug MLN0128, which inhibits both complexes of mTOR (mTORC1 and mTORC2), and the hydroxamic acid pan-HDAC inhibitor TSA synergistically inhibit the viability of a phenotypically diverse panel of five breast cancer cell lines (HR-/+, HER2-/+). The combination of MLN0128 and TSA induces apoptosis in most breast cancer cell lines tested, but not in the non-malignant MCF-10A mammary epithelial cells. In parallel, the MLN0128/TSA combination reduces phosphorylation of AKT at S473 more than single agents alone and more so in the 5 malignant breast cancer cell lines than in the non-malignant mammary epithelial cells. Examining polysome profiles from one of the most sensitive breast cancer cell lines (SKBR3), we demonstrate that this MLN0128/TSA treatment combination synergistically impairs polysome assembly in conjunction with enhanced inhibition of 4eBP1 phosphorylation at S65. Taken together, these data indicate that the synergistic growth inhibiting consequence of combining a mTORC1/C2 inhibitor like MLN0128 with a pan-HDAC inhibitor like TSA results from their mechanistic convergence onto the PI3K/AKT/mTOR pathway, profoundly inhibiting both AKT S473 and 4eBP1 S65 phosphorylation, reducing polysome formation and cancer cell viability.

First pregnancy events and future breast density: modification by age at first pregnancy and specific VEGF and IGF1R gene variants.

PURPOSE: Pregnancy characteristics have been associated with breast cancer risk, but information is limited on their relationship with breast density. Our objective was to examine the relationship between first pregnancy characteristics and later life breast density, and whether the association is modified by genotype. METHODS: The Marin Women's Study was initiated to examine breast cancer in a high-incidence mammography population (Marin County, CA). Reproductive characteristics and pregnancy information including pregnancy-induced hypertension (PIH) were self-reported at the time of mammography. Forty-seven candidate single nucleotide polymorphisms were obtained from saliva samples; seven were assessed in relation to PIH and percent fibroglandular volume (%FGV). Breast density assessed as %FGV was measured on full-field digital mammograms by the San Francisco Mammography Registry. RESULTS: A multivariable regression model including 2,440 parous women showed that PIH during first pregnancy was associated with a statistically significant decrease in %FGV (b = -0.31, 95 % CI -0.52, -0.11), while each month of breast-feeding after first birth was associated with a statistically significant increase in %FGV (b = 0.01, 95% CI 0.003, 0.02). PIH and breast-feeding associations with %FGV were modified by age at first birth. In a subsample of 1,240 women, there was evidence of modification in the association between PIH and %FGV by specific vascular endothelial growth factor (VEGF) (rs3025039) and insulin growth factor receptor-1 (IGFR1) (rs2016347) gene variants. CONCLUSION: These findings suggest that first pregnancy characteristics may exert an influence on extent of breast density later in life and that this influence may vary depending on inherited IGFR1 and VEGF genotypes.

Brain transcriptomic, metabolic and mitohormesis properties associated with N-propargylglycine treatment: A prevention strategy against neurodegeneration.

INTRODUCTION: There is an urgent need for new or repurposed therapeutics that protect against or significantly delay the clinical progression of neurodegenerative diseases, such as Huntington's disease (HD), Parkinson's disease and Alzheimer's disease. In particular, preclinical studies are needed for well tolerated and brain-penetrating small molecules capable of mitigating the proteotoxic mitochondrial processes that are hallmarks of these diseases. We identified a unique suicide inhibitor of mitochondrial proline dehydrogenase (Prodh), N-propargylglycine (N-PPG), which has anticancer and brain-enhancing mitohormesis properties, and we hypothesize that induction of mitohormesis by N-PPG protects against neurodegenerative diseases. We carried out a series of mouse studies designed to: i) compare brain and metabolic responses while on oral N-PPG treatment (50 mg/kg, 9-14 days) of B6CBA wildtype (WT) and short-lived transgenic R6/2 (HD) mice; and ii) evaluate potential brain and systemwide stress rebound responses in WT mice 2 months after cessation of extended mitohormesis induction by well-tolerated higher doses of N-PPG (100-200 mg/kg x 60 days). WT and HD mice showed comparable global evidence of N-PPG induced brain mitohormesis characterized by Prodh protein decay and increased mitochondrial expression of chaperone and Yme1l1 protease proteins. Interestingly, transcriptional analysis (RNAseq) showed partial normalization of HD whole brain transcriptomes toward those of WT mice. Comprehensive metabolomic profiles performed on control and N-PPG treated blood, brain, and kidney samples revealed expected N-PPG-induced tissue increases in proline levels in both WT and HD mice, accompanied by surprising parallel increases in hydroxyproline and sarcosine. Two months after cessation of the higher dose N-PPG stress treatments, WT mouse brains showed robust rebound increases in Prodh protein levels and mitochondrial transcriptome responses, as well as altered profiles of blood amino acid-related metabolites. Our HD and WT mouse preclinical findings point to the brain penetrating and mitohormesis-inducing potential of the drug candidate, N-PPG, and provide new rationale and application insights supporting its further preclinical testing in various models of neurodegenerative diseases characterized by loss of mitochondrial proteostasis.

Therapeutic targeting of HYPDH/PRODH2 with N-propargylglycine offers a Hyperoxaluria treatment opportunity.

N-propargylglycine prevents 4-hydroxyproline catabolism in mouse liver and kidney. N-propargylglycine is a novel suicide inhibitor of PRODH2 and induces mitochondrial degradation of PRODH2. PRODH2 is selectively expressed in liver and kidney and contributes to primary hyperoxaluria (PH). Preclinical evaluation of N-propargylglycine efficacy as a new PH therapeutic is warranted.

An optimized five-gene multi-platform predictor of hormone receptor negative and triple negative breast cancer metastatic risk.

INTRODUCTION: Outcome predictors in use today are prognostic only for hormone receptor-positive (HRpos) breast cancer. Although microarray-derived multigene predictors of hormone receptor-negative (HRneg) and/or triple negative (Tneg) breast cancer recurrence risk are emerging, to date none have been transferred to clinically suitable assay platforms (for example, RT-PCR) or validated against formalin-fixed paraffin-embedded (FFPE) HRneg/Tneg samples. METHODS: Multiplexed RT-PCR was used to assay two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4) in a pooled FFPE collection of 139 chemotherapy-naïve HRneg breast cancers. The prognostic value of the RTPCR measured gene signatures were evaluated as continuous and dichotomous variables, and in conditional risk models incorporating clinical parameters. An optimized five-gene index was derived by evaluating gene combinations from both signatures. RESULTS: RT-PCR measured IR-7 and Buck-4 signatures proved prognostic as continuous variables; and conditional risk modeling chose nodal status, the IR-7 signature, and tumor grade as significant predictors of distant recurrence (DR). From the Buck-4 and IR-7 signatures, an optimized five-gene (TNFRSF17, CLIC5, HLA-F, CXCL13, XCL2) predictor was generated, referred to as the Integrated Cytokine Score (ICS) based on its functional pathway linkage through interferon-γ and IL-10. Across all FFPE cases, the ICS was prognostic as either a continuous or dichotomous variable, and conditional risk modeling selected nodal status and ICS as DR predictors. Further dichotomization of node-negative/ICS-low FFPE cases identified a subset of low-grade HRneg tumors with <10% 5-year DR risk. The prognostic value of ICS was reaffirmed in two previously studied microarray assayed cohorts containing 274 node-negative and chemotherapy naive HRneg breast cancers, including 95 Tneg cases where it proved prognostically independent of Tneg molecular subtyping. In additional HRneg/Tneg microarray assayed cohorts, the five-gene ICS also proved prognostic irrespective of primary tumor nodal status and adjuvant chemotherapy intervention. CONCLUSION: We advanced the measurement of two previously reported microarray-derived HRneg/Tneg breast cancer prognostic signatures for use in FFPE samples, and derived an optimized five-gene Integrated Cytokine Score (ICS) with multi-platform capability of predicting metastatic outcome from primary HRneg/Tneg tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype.

FOXP3-positive regulatory T lymphocytes and epithelial FOXP3 expression in synchronous normal, ductal carcinoma in situ, and invasive cancer of the breast.

FOXP3-expressing T regulatory lymphocytes (Tregs) have been described as putative mediators of immune tolerance, and thus facilitators of tumor growth. When found in association with various malignancies, Tregs are generally markers of poor clinical outcome. However, it is unknown whether they are also associated with cancer progression. We evaluated quantitative FOXP3 expression in lymphocytes as well as in epithelial cells in a set of thirty-two breast tumors with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Tumors were stained for FOXP3 and CD3 expression and Tregs quantified by determining the ratio of colocalized FOXP3 and CD3 relative to 1) total CD3-expressing lymphocytes and 2) to FOXP3-expressing epithelial cells. The median proportion of FOXP3-expressing CD3 cells significantly increased with malignant progression from normal to DCIS to IDC components (0.005, 0.019 and 0.030, respectively; p ≤ 0.0001 for normal vs. IDC and p = 0.004 for DCIS vs. IDC). The median intensity of epithelial FOXP3 expression was also increased with invasive progression and most markedly augmented between normal and DCIS components (0.130 vs. 0.175, p ≤ 0.0001). Both Treg infiltration and epithelial FOXP3 expression were higher in grade 3 vs. grade 1 tumors (p = 0.014 for Tregs, p = 0.038 for epithelial FOXP3), but did not vary significantly with hormone receptor status, size of invasive tumor, lymph node status, or disease stage. Notably, Treg infiltration significantly correlated with epithelial up-regulation of FOXP3 expression (p = 0.013 for normal, p = 0.001 for IDC). These findings implicate both Treg infiltration and up-regulated epithelial FOXP3 expression in breast cancer progression.