Black heterosexual men's resilience in times of HIV adversity: findings from the "weSpeak" study.

BACKGROUND: In Canada, heterosexual African, Caribbean and Black (ACB) men tend to suffer a disproportionate burden of HIV. Consequently, studies have examined the underlying contributors to this disparity through the nexus of behavioral and structural factors. While findings from these studies have been helpful, their use of deficit and risk models only furthers our knowledge of why ACB men are more vulnerable to HIV infection. Thus far, there is a dearth of knowledge on how heterosexual ACB men mobilize protective assets to promote their resilience against HIV infection. METHODS: As part of a larger Ontario-based project called weSpeak, this study examined how ACB men acquire protective assets to build their resilience to reduce their HIV vulnerability. We analyzed three focus group discussions (n = 17) and 13 in-depth interviews conducted with ACB men using NVivo and a mixed inductive-deductive thematic analyses approach. RESULTS: The findings show that ACB men mostly relied on personal coping strategies, including sexual abstinence, to build resilience against HIV. Interpersonal resources such as family, friends, and religious communities also played an important role in constructing ACB men's resilience. ACB men bemoaned their lack of access to essential institutional resources, such as health services, that are important in managing HIV adversity. CONCLUSION: Based on these findings, there is an urgent need for HIV policy stakeholders, including service providers, to engage the ACB community in the design of intervention programs. Additionally, addressing the socioeconomic disadvantages faced by ACB communities will increase the capacity of ACB men to develop resilience against HIV.

Knowledge of sexual partner's HIV serostatus and the practice of safer sex among heterosexual men of African descent in London, Ontario.

Objective: Poor knowledge of sexual partners' HIV status is a major contributing factor in the heterosexual spread of HIV in Canada. This study examined knowledge of sexual partner's HIV serostatus and the practice of safer sex among self-identified heterosexual African, Caribbean and Black (ACB) men in London, Ontario.Design: A cross-sectional data was collected from 156 heterosexual ACB men in London. The negative log-log link function was fitted to estimate the relationship between knowledge of sexual partner's HIV status and condom use among ACB men.Results: Findings show that ACB men who know their sexual partner's HIV status are less likely to use condoms compared to men who do not know the serostatus of their sexual partner, controlling for other theoretically relevant covariates. In addition, the findings show that sexually active, single ACB men are less likely to use condoms. On the other hand, ACB men with higher education, employed and with income over 60 thousand dollars a year have a higher likelihood of using condoms.Conclusions: Heterosexual ACB men who used condoms even when they did not know their sexual partners' HIV status could be explained as a resilience-building strategy in response to their increasing HIV vulnerabilities. Heterosexual ACB men's use of condoms is further associated with socioeconomic factors including income, employment and education that need to be addressed for an improved safer sex.

Sexual Debut Among Heterosexual Men of African and Caribbean Descent: Are the Youth Initiating Sex Earlier than the Older Generation?

Although age at first sex is considered a measure of sexual risk and vulnerability for HIV infection, there is a dearth of literature on age at sexual debut in the Canadian context. This study examined time variations to first sex among heterosexual African, Caribbean, and Black (ACB) men in four Ontarian cities. A population-based retrospective survey (n = 879) on timing to first sexual intercourse was conducted between 2018 and 2019 among self-identified heterosexual ACB men 16 years or older and residing in London, Ottawa, Toronto or Windsor. We used the lognormal survival analysis technique to examine variations in time to first sexual intercourse among age cohorts and between cities. The findings showed a generational shift in the pattern of sexual initiation, with younger heterosexual ACB men initiating sexual intercourse earlier compared with those currently older than 50 years. We observed those between 16 and 19 years, 20 and 29 years, and 30 and 39 years of age to have significantly higher risk ratios of TR = 0.852, TR = 0.869, and TR = 0.855, respectively. At city level, the results show marked spatial variations, with youth in cities of Toronto, Ottawa, and London at the highest risk of early sexual debut relative to those in Windsor. Early initiation of first sexual intercourse among heterosexual ACB youth was observed with those in the larger cities being at a relatively higher risk. There is the need for programs aimed at delaying sexual debut among youth in general. It is, however, important to recognize the relative risk of those in the larger cities.

"I went in there, had a bit of an issue with those folks": everyday challenges of heterosexual African, Caribbean and black (ACB) men in accessing HIV/AIDS services in London, Ontario.

BACKGROUND: In Canada, heterosexual African, Caribbean, and Black (ACB) men's heightened risk of HIV infection has been linked to behavioral characteristics, including practices of hegemonic masculinity that discourage the use of HIV preventive services. However, this framing is bereft of the role of structural factors that may be contributing to new HIV infections. This paper examined the underlying factors limiting access to health services among heterosexual ACB men in London, Ontario Canada. METHODS: A convenient sampling technique was used to recruit thirty-seven (n = 37) self-identified heterosexual ACB men and service providers. Four focus groups (FG) were conducted; three with ACB participants of similar age category (i.e., 16-24; 25-38; 39+), and one with service providers. The FGs focused on the barriers to using health services and interrogated the ease of access to HIV intervention programs by ACB men respectively. Recurring themes from the FGs were probed further using in-depth interviews (n = 13). FGs and in-depth interviews complemented each other in reducing uneven power dynamics, fact checking, and allowing for detail discussion of the topic under study. Data analyses were done in NVivo using a mixed inductive-deductive thematic analyses approach. RESULTS: Most ACB men lacked information on HIV and were unaware of their increased risk of infection. Contrary to the notion that behavioral characteristics keep ACB men away from health services, we found that most ACB men were unaware of the availability of these services. Those that had some knowledge about the services reported that they were not appropriately tailored to their needs. In addition, stereotypes and stigma about the etiology of HIV among Blacks, and systemic neglect served as significant barriers to ACB men's use of services. CONCLUSION: The findings suggest that, to enhance preventive health service use among heterosexual ACB men, there is the need to remove structural barriers. Engaging ACB men in the design and implementation of policies may be useful at improving access to HIV information, testing, and treatment services. Increased information dissemination to ACB men would create awareness of the availability of HIV services. Finally, service providers should be conscious of ACB men's concern about experiences of discrimination and racism at service centers.

An Intersectional Approach to HIV Vulnerabilities and Testing Among Heterosexual African Caribbean and Black Men in London, Ontario: Results From the weSpeak Study.

Heterosexual African, Caribbean and Black (ACB) men are a vulnerable group to HIV infection in Canada, but little is known about their uptake of HIV testing services. Studies on ACB men HIV vulnerabilities have largely focused on behavioural factors. While these studies have contributed to the current HIV prevention success in Canada, little attention has been paid to structural factors that intersect with prevailing behaviours to reinforce vulnerabilities. Drawing insights from intersectionality theory, we examined healthcare access and HIV testing among heterosexual ACB men in London, Ontario. We fitted the negative log-log link function to 155 individuals' survey. Results show that participants, who had difficulty accessing healthcare, experienced discrimination, and were young, were all less likely to test for HIV. Even though the probability of testing for HIV increased after accounting for the effect of structural factors, the marginal impact was higher for those without any difficulty accessing healthcare than those with difficulty. Findings are discussed within the broader theory of intersectionality and recommendations made for public health policy.

Tumor-infiltrating BRAFV600E-specific CD4+ T cells correlated with complete clinical response in melanoma.

T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who achieved a complete response following adoptive transfer of tumor-infiltrating lymphocytes (TILs). Tumor exome sequencing surprisingly revealed fewer than 30 nonsynonymous somatic mutations, including oncogenic BRAFV600E. Analysis of the specificity of TILs identified rare CD4+ T cells specific for BRAFV600E and diverse CD8+ T cells reactive to nonmutated self-antigens. These specificities increased in blood after TIL transfer and persisted long-term, suggesting they contributed to the effective antitumor immune response. Gene transfer of the BRAFV600E-specific T cell receptor (TCR) conferred recognition of class II MHC-positive cells expressing the BRAF mutation. Therapy with TCR-engineered BRAFV600E-specific CD4+ T cells may have direct antitumor effects and augment CD8+ T cell responses to self- and/or mutated tumor antigens in patients with BRAF-mutated cancers.

Identifying Abundant Immunotherapy and Other Targets in Solid Tumors: Integrating RNA-seq and Mass Spectrometry Proteomics Data Sets.

RNA-seq and mass-spectrometry proteomics combined with growing data repositories have greatly increased the capacity to identify candidate proteins or protein sequence variants that share properties of ideal therapy targets, which include being abundant in cancer cells, absent or rare in adult organs (especially vital organs), and shared by many patient tumors. RNA-seq and fixed content arrays can identify genes that are overexpressed or misexpressed in cancer. RNA-seq is uniquely suited to identifying cancer-specific sequence variants. We review factors relevant for determining whether products of genes that are abundant or differentially abundant in RNA-seq are concordant or discordant with proteins that are identified as abundant or differentially abundant in mass-spectrometry proteomics assays.

Neutrophils dominate the immune cell composition in non-small cell lung cancer.

The response rate to immune checkpoint inhibitor therapy for non-small-cell lung cancer (NSCLC) is just 20%. To improve this figure, several early phase clinical trials combining novel immunotherapeutics with immune checkpoint blockade have been initiated. Unfortunately, these trials have been designed without a strong foundational knowledge of the immune landscape present in NSCLC. Here, we use a flow cytometry panel capable of measuring 51 immune cell populations to comprehensively identify the immune cell composition and function in NSCLC. The results show that the immune cell composition is fundamentally different in lung adenocarcinoma as compared with lung squamous cell carcinoma, and that neutrophils are the most prevalent immune cell type. Using T-cell receptor-ß sequencing and tumour reactivity assays, we predict that tumour reactive T cells are frequently present in NSCLC. These results should help to guide the design of clinical trials and the direction of future research in this area.

Tumor-Infiltrating Merkel Cell Polyomavirus-Specific T Cells Are Diverse and Associated with Improved Patient Survival.

Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer+ CD8+ T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRß (TRB) sequencing was performed on tetramer+ cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer+ T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137-47. ©2017 AACR.

Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia.

With the demonstration of improved survival of some acute myeloid leukemia (AML) patients with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), CD33 has been validated as a target for antigen-specific immunotherapy. Since previous studies identified a CD33 splice variant missing exon 2 (CD33∆E2) and, consequently, the immune-dominant membrane-distal V-set domain, we investigated the expression and functional characteristics of CD33 transcript variants in AML. In primary AML specimens, we not only found full-length CD33 (CD33FL) and CD33∆E2 but also corresponding variants containing an alternate exon 7 predicted to encode a CD33 protein lacking most of the intracellular domain (CD33E7a and, not previously described, CD33∆E2,E7a) in almost all cases. In acute leukemia cell sublines engineered to express individual CD33 splice variants, all splice variants had endocytic properties. CD33FL and CD33E7a mediated similar degrees of GO cytotoxicity, whereas CD33∆E2 and CD33∆E2,E7a could not serve as target for GO. Co-expression of CD33∆E2 did not interfere with CD33FL endocytosis and did not impact CD33FL-mediated GO cytotoxicity. Together, our findings document a greater-than-previously thought complexity of CD33 expression in human AML. They identify CD33 variants that lack exon 2 and are not recognized by current CD33-directed therapeutics as potential target for future unconjugated or conjugated antibodies.

Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFß-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that 'High-RPS8 and Low-PRELP' was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated.

Quantitative glycoproteomics analysis reveals changes in N-glycosylation level associated with pancreatic ductal adenocarcinoma.

Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-ß, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events.

Phenotypic and transcriptional fidelity of patient-derived colon cancer xenografts in immune-deficient mice.

Xenografts of human colorectal cancer (CRC) in immune-deficient mice have great potential for accelerating the study of tumor biology and therapy. We evaluated xenografts established in NOD/scid/IL2Rγ-null mice from the primary or metastatic tumors of 27 patients with CRC to estimate their capacity for expanding tumor cells for in vitro studies and to assess how faithfully they recapitulated the transcriptional profile of their parental tumors. RNA-seq analysis of parental human CRC tumors and their derivative xenografts demonstrated that reproducible transcriptional changes characterize the human tumor to murine xenograft transition. In most but not all cases, the human stroma, vasculature, and hematopoietic elements were systematically replaced by murine analogues while the carcinoma component persisted. Once established as xenografts, human CRC cells that could be propagated by serial transplantation remained transcriptionally stable. Three histologically atypical xenografts, established from patients with peritoneal metastases, contained abundant human stromal elements and blood vessels in addition to human tumor cells. The transcriptomes of these mixed tumor/stromal xenografts did not closely resemble those of their parental tumors, and attempts to propagate such xenografts by serial transplantation were unsuccessful. Stable expression of numerous genes previously identified as high priority targets for immunotherapy was observed in most xenograft lineages. Aberrant expression in CRC cells of human genes that are normally only expressed in hematopoietic cells was also observed. Our results suggest that human CRC cells expanded in murine xenografts have great utility for studies of tumor immunobiology and targeted therapies such as immunotherapy but also identify potential limitations.

Metabolomic profiling of urine: response to a randomised, controlled feeding study of select fruits and vegetables, and application to an observational study.

Metabolomic profiles were used to characterise the effects of consuming a high-phytochemical diet compared with a diet devoid of fruits and vegetables (F&V) in a randomised trial and cross-sectional study. In the trial, 8 h fasting urine from healthy men (n 5) and women (n 5) was collected after a 2-week randomised, controlled trial of two diet periods: a diet rich in cruciferous vegetables, citrus and soya (F&V), and a fruit- and vegetable-free (basal) diet. Among the ions found to differentiate the diets, 176 were putatively annotated with compound identifications, with forty-six supported by MS/MS fragment evidence. Metabolites more abundant in the F&V diet included markers of the dietary intervention (e.g. crucifers, citrus and soya), fatty acids and niacin metabolites. Ions more abundant in the basal diet included riboflavin, several acylcarnitines and amino acid metabolites. In the cross-sectional study, we compared the participants based on the tertiles of crucifers, citrus and soya from 3 d food records (n 36) and FFQ (n 57); intake was separately divided into the tertiles of total fruit and vegetable intake for FFQ. As a group, ions individually differential between the experimental diets differentiated the observational study participants. However, only four ions were significant individually, differentiating the third v. first tertile of crucifer, citrus and soya intake based on 3 d food records. One of these ions was putatively annotated: proline betaine, a marker of citrus consumption. There were no ions significantly distinguishing tertiles by FFQ. The metabolomic assessment of controlled dietary interventions provides a more accurate and stronger characterisation of the diet than observational data.

Significance of expression of ITGA5 and its splice variants in acute myeloid leukemia: a report from the Children's Oncology Group.

Acute myeloid leukemia (AML) encompasses a heterogeneous group of diseases, and novel biomarkers for risk refinement and stratification are needed to optimize patient care. To identify novel risk factors, we performed transcriptome sequencing on 68 diagnostic AML samples and identified 2 transcript variants (-E2 and -E2/3) of the α-subunit (ITGA5) of the very late antigen-5 integrin. We then quantified expression of ITGA5 and these splice variants in specimens from participants of the AAML03P1 trial. We found no association between ITGA5 expression and clinical outcome. In contrast, patients with the highest relative expression (Q4) of the -E2/3 ITGA5 splice variant less likely had low-risk disease than Q1-3 patients (21% vs. 38%, P = 0.027). Q4 patients had worse response to chemotherapy with a higher proportion having persistent minimal residual disease (50% vs. 23%, P = 0.003) and inferior overall survival (at 5 years: 48% vs. 67%, P = 0.015); the latter association was limited to low-risk patients (Q4 vs. Q1-3: 56% vs. 85%, P = 0.043) and was not seen in standard-risk (51% vs. 60%, P = 0.340) or high-risk (33% vs. 38%, P = 0.952) patients. Our exploratory studies indicate that transcriptome sequencing is useful for biomarker discovery, as exemplified by the identification of ITGA5 -E2/3 splice variant as potential novel adverse prognostic marker for low-risk AML that, if confirmed, could serve to further risk-stratify this patient subset.

Autoantibody signatures involving glycolysis and splicesome proteins precede a diagnosis of breast cancer among postmenopausal women.

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen-antibody interactions.

Statistical inference from multiple iTRAQ experiments without using common reference standards.

Isobaric tags for relative and absolute quantitation (iTRAQ) is a prominent mass spectrometry technology for protein identification and quantification that is capable of analyzing multiple samples in a single experiment. Frequently, iTRAQ experiments are carried out using an aliquot from a pool of all samples, or "masterpool", in one of the channels as a reference sample standard to estimate protein relative abundances in the biological samples and to combine abundance estimates from multiple experiments. In this manuscript, we show that using a masterpool is counterproductive. We obtain more precise estimates of protein relative abundance by using the available biological data instead of the masterpool and do not need to occupy a channel that could otherwise be used for another biological sample. In addition, we introduce a simple statistical method to associate proteomic data from multiple iTRAQ experiments with a numeric response and show that this approach is more powerful than the conventionally employed masterpool-based approach. We illustrate our methods using data from four replicate iTRAQ experiments on aliquots of the same pool of plasma samples and from a 406-sample project designed to identify plasma proteins that covary with nutrient concentrations in chronically undernourished children from South Asia.

Longitudinal screening algorithm that incorporates change over time in CA125 levels identifies ovarian cancer earlier than a single-threshold rule.

PURPOSE: Longitudinal algorithms incorporate change over time in biomarker levels to individualize screening decision rules. Compared with a single-threshold (ST) rule, smaller deviations from baseline biomarker levels are required to signal disease. We demonstrated improvement in ovarian cancer early detection by using a longitudinal algorithm to monitor annual CA125 levels. PATIENTS AND METHODS: We retrospectively evaluated serial preclinical serum CA125 values measured annually in 44 incident ovarian cancer cases identified from participants in the PLCO (Prostate Lung Colorectal and Ovarian) Cancer Screening Trial to determine how frequently and to what extent the parametric empirical Bayes (PEB) longitudinal screening algorithm identifies ovarian cancer earlier than an ST rule. RESULTS: The PEB algorithm detected ovarian cancer earlier than an ST rule in a substantial proportion of cases. At 99% specificity, which corresponded to the ST-rule CA125 cutoff ≥ 35 U/mL that was used in the PLCO trial, 20% of cases were identified earlier by using the PEB algorithm. Among these cases, the PEB signaled abnormal CA125 values, on average, 10 months earlier and at a CA125 concentration 42% lower (20 U/mL) than the ST-rule cutoff. The proportion of cases detected earlier by the PEB algorithm and the earliness of detection increased as the specificity of the screening rule was reduced. CONCLUSION: The PEB longitudinal algorithm identifies ovarian cancer earlier and at lower biomarker concentrations than an ST screening algorithm adjusted to the same specificity. Longitudinal biomarker assessment by using the PEB algorithm may have application for screening other solid tumors in which biomarkers are available.

Interpretation of single and serial measures of HE4 and CA125 in asymptomatic women at high risk for ovarian cancer.

BACKGROUND: Human epididymis protein 4 (HE4) is approved for clinical use with CA125 to predict epithelial ovarian cancer in women with a pelvic mass or in remission after chemotherapy. Previously reported reference ranges for HE4 are inconsistent. METHODS: We report positivity thresholds yielding 90%, 95%, 98%, and 99% specificity for age-defined populations of healthy women for HE4, CA125, and Risk of Ovarian Malignancy Algorithm (ROMA), a weighted average of HE4 and CA125. HE4 and CA125 were measured in 1,780 samples from 778 healthy women aged >25 years with a documented deleterious mutation, or aged >35 years with a significant family history. Effects on marker levels of a woman's age, ethnicity, and epidemiologic characteristics were estimated, as were the population-specific means, variances, and within- and between-woman variances used to generate longitudinal screening algorithms for these markers. RESULTS: CA125 levels were lower with Black ethnicity (P = 0.008). Smoking was associated with higher HE4 (P = 0.007) and ROMA (P < 0.019). Continuous oral contraceptive use decreased levels of CA125 (P = 0.041), and ROMA (P = 0.12). CA125 was lower in women age ≥55, and HE4 increased with age (P < 0.01), particularly among women age ≥55. CONCLUSIONS: Because of the strong effect of age on HE4, thresholds for HE4 are best defined for women of specific ages. Age-specific population thresholds for HE4 for 95% specificity ranged from 41.4 pmol/L for women age 30 to 82.1 pmol/L for women age 80. IMPACT: Incorporation of serial marker values from screening history reduces personalized thresholds for CA125 and HE4 but is inappropriate for ROMA.