Jonathan L. Hecht

Lifetime Exposure to Cigarette Smoke, B-Cell Tumor Immune Infiltration, and Immunoglobulin Abundance in Ovarian Tumors

Abstract Background: Cigarette smoke exposure has been linked to systemic immune dysfunction, including for B-cell and immunoglobulin (Ig) production, and poor outcomes in patients with ovarian cancer. No study has evaluated the impact of smoke exposure across the life-course on B-cell infiltration and Ig abundance in ovarian tumors. Methods: We measured markers of B and plasma cells and Ig isotypes using multiplex immunofluorescence on 395 ovarian cancer tumors in the Nurses’ Health Study (NHS)/NHSII. We conducted beta-binomial analyses evaluating odds ratios (OR) and 95% confidence intervals (CI) for positivity of immune markers by cigarette exposure among cases and Cox proportional hazards models to evaluate hazard ratios (HR) and 95% CI for developing tumors with low (<median) or high (≥median) immune cell/Ig percentage. Results: There were no associations between smoke exposure and B-cell or IgM infiltration in ovarian tumors. Among cases, we observed higher odds of IgA+ among ever smokers (OR, 1.54; 95% CI, 1.14–2.07) and ever smokers with no parental smoke exposure (OR, 2.03; 95% CI, 1.18–3.49) versus never smokers. Women with parental cigarette smoke exposure versus not had higher risk of developing ovarian cancer with low IgG+ (HR, 1.51; 95% CI, 1.10–2.09), whereas ever versus never smokers had a lower risk (HR, 0.74; 95% CI, 0.56–0.99). Conclusions: Ever smoking was associated with increased odds of IgA in ovarian tumors. Impact: IgA has been associated with improved ovarian cancer outcomes, suggesting that although smoking is associated with poor outcomes in patients with ovarian cancer, it may lead to improved tumor immunogenicity.

Measurement of Ovarian Tumor Immune Profiles by Multiplex Immunohistochemistry: Implications for Epidemiologic Studies

Abstract Background: Despite the immunogenic nature of many ovarian tumors, treatment with immune checkpoint therapies has not led to substantial improvements in ovarian cancer survival. To advance population-level research on the ovarian tumor immune microenvironment, it is critical to understand methodologic issues related to measurement of immune cells on tissue microarrays (TMA) using multiplex immunofluorescence (mIF) assays. Methods: In two prospective cohorts, we collected formalin-fixed, paraffin-embedded ovarian tumors from 486 cases and created seven TMAs. We measured T cells, including several sub-populations, and immune checkpoint markers on the TMAs using two mIF panels. We used Spearman correlations, Fisher exact tests, and multivariable-adjusted beta-binomial models to evaluate factors related to immune cell measurements in TMA tumor cores. Results: Between-core correlations of intratumoral immune markers ranged from 0.52 to 0.72, with more common markers (e.g., CD3+, CD3+CD8+) having higher correlations. Correlations of immune cell markers between the whole core, tumor area, and stromal area were high (range 0.69–0.97). In multivariable-adjusted models, odds of T-cell positivity were lower in clear cell and mucinous versus type II tumors (ORs, 0.13–0.48) and, for several sub-populations, were lower in older tissue (sample age > 30 versus ≤ 10 years; OR, 0.11–0.32). Conclusions: Overall, high correlations between cores for immune markers measured via mIF support the use of TMAs in studying ovarian tumor immune infiltration, although very old samples may have reduced antigenicity. Impact: Future epidemiologic studies should evaluate differences in the tumor immune response by histotype and identify modifiable factors that may alter the tumor immune microenvironment.

Estrogen Receptor-β Expression of Ovarian Tumors and Its Association with Ovarian Cancer Risk Factors

Abstract Background: Differential associations between ovarian cancer risk factors and estrogen receptor-α (ERα) ovarian tumor expression have been noted; however, no research has assessed estrogen receptor-β (ERβ) expression. Thus, in exploratory analyses, we assessed the association of several factors with ovarian cancer risk by ERβ tumor status. Methods: We conducted a nested case–control study within the prospective Nurses' Health Study cohorts (NHS/NHSII), with exposures collected through biennial questionnaires. Paraffin-embedded tumor blocks were requested for cases diagnosed from 1976 to 2006 (NHS) and 1989 to 2005 (NHSII) and tissue microarrays were stained for nuclear ERβ (ERβ-nuc) and cytoplasmic ERβ (ERβ-cyto), with any staining considered positive (+). We obtained odds ratios (OR) and 95% confidence intervals (CI) using multivariate polytomous logistic regression. Results: We included 245 cases [43% ERβ-cyto (+) and 71% ERβ-nuc (+)] and 1,050 matched controls. An inverse association was observed between parity and risk of ERβ-nuc (+) (OR, parous vs. nulliparous: 0.46; 95% CI, 0.26–0.81), but not ERβ-nuc (–) tumors (OR, parous vs. nulliparous: 1.51; 95% CI, 0.45–5.04; Pheterogeneity = 0.04). Conversely, parity was inversely associated with ERβ-cyto (–) tumors (OR, parous vs. nulliparous: 0.42; 95% CI, 0.23–0.78), but was not associated with ERβ-cyto (+) tumors (OR, parous vs. nulliparous: 1.08; 95% CI, 0.45–2.63; Pheterogeneity = 0.05). Associations for other exposures, including hormone therapy, did not differ by ERβ-nuc or ERβ-cyto status. Conclusions: Our results suggest that parity may influence ovarian cancer risk, in part, through alterations in ERβ localization within tumor cells. Impact: Alterations in ERβ expression and localization appear to be important for ovarian cancer etiology. Future research should confirm our results and assess potential biologic mechanisms for the observed associations.

Prospective Analyses of Lifestyle Factors Related to Energy Balance and Ovarian Cancer Risk by Infiltration of Tumor-Associated Macrophages

Abstract Background: Lifestyle factors related to energy balance have been associated with ovarian cancer risk and influence the tumor immune microenvironment, including tumor-associated macrophages (TAM). However, no studies have assessed whether these factors differentially impact ovarian cancer risk by TAM densities. Methods: We conducted a prospective analysis in the Nurses' Health Studies to examine the associations of physical activity, sitting time, and a food-based empirical dietary inflammatory pattern (EDIP) score with invasive epithelial ovarian cancer risk by TAM density assessed by immunohistochemistry. We considered density of CD68 (marker of total TAMs) and CD163 (marker of pro-carcinogenic M2-type TAMs), and their ratios. We used multivariable Cox proportional hazards regression to calculate hazard ratios (HR) and 95% confidence intervals (CI) of exposures with risk of ovarian tumors with high versus low TAMs, including analyses stratified by body mass index. Results: Analyses included 312 incident ovarian cancer cases with TAM measurements. Physical activity, sitting time, and EDIP score were not differentially associated with ovarian cancer risk by TAM densities (Pheterogeneity > 0.05). Among overweight and obese women, higher EDIP score was associated with increased risk of CD163 low-density tumors (HR comparing extreme tertiles, 1.57; 95% CI, 0.88–2.80; Ptrend = 0.01), but not CD163 high-density tumors (comparable HR, 1.16; 95% CI, 0.73–1.86; Ptrend = 0.24), though this difference was not statistically significant (Pheterogeneity = 0.22). Conclusions: We did not observe differential associations between lifestyle factors and ovarian cancer risk by TAM densities. Impact: Future investigations examining the interplay between other ovarian cancer risk factors and the tumor immune microenvironment may help provide insight into ovarian cancer etiology.

Impact of a Modified HistoGel Method for Processing Endocervical Curettage Specimens on Diagnostic Yield

Abstract Objectives Endocervical curettage (ECC) specimens may be limited by scant tissue. We evaluated whether a cellular concentration processing method could improve their diagnostic quality. Methods Between October 2018 and June 2019, ECC specimens were assigned chronologically to one of two groups: nonconcentrated ECC (NECC) or concentrated ECC (CECC). NECC specimens underwent routine histologic processing. CECC specimens were processed using a published HistoGel-based cell block method. We reviewed diagnoses for ECCs, concurrent cervical biopsies and/or loop electrosurgical excision procedures (LEEPs), and preceding Papanicolaou (Pap) smears. We performed multivariate logistic regression analyses to evaluate the impact of processing method on ECC adequacy and discordance between Pap smear and worst tissue diagnoses. Results NECC and CECC adequacy was 88.2% and 84.7% (P = .06). ECC adequacy was greater if concurrent biopsy/LEEP was performed (odds ratio [OR] = 1.76, P < .01). Discordance between Pap smear and worst tissue diagnoses was 9.5% and 13.3% (P = .04) for cases with NECC and CECC processing, although processing method was not significant in multivariate analysis (OR = 0.74, P = .11). Adequate ECC sampling and concurrent biopsy/LEEP were independently associated with concordance between Pap smear and worst tissue diagnosis (OR = 0.46, P < .01 and OR = 0.65, P = .02). Conclusions ECC processing method did not significantly affect either specimen adequacy (P = .06) or diagnostic discordance (P = .11) when controlled for other factors.

Associations between Parity, History of Breastfeeding, and T-cell Profile of Ovarian Tumors

Abstract Background: Parity and breastfeeding are associated with systemic changes in maternal inflammation and reduced risk of ovarian cancer, but little is known about their impact on the ovarian tumor immune microenvironment. Methods: We evaluated the associations of self-reported parity and history of breastfeeding with tumor-infiltrating T cells among 1,706 ovarian carcinoma cases with tumor tissue collected across four studies. The abundance of tumor-infiltrating T cells was measured by multiplex immunofluorescence in tumor tissue microarrays. ORs and 95% confidence intervals (CI) for the positivity of tumor immune cells were calculated using beta-binomial models and stratified by histotype. Results: Compared with ovarian tumors in nulliparous women, there was no association between parity and ovarian tumor T-cell abundance among all histotypes combined but suggestion of increased cytotoxic T cells and T-cell exhaustion among parous women with clear-cell tumors. When restricted to parous women, history of breastfeeding was associated with increased odds for all T-cell types [i.e., total T, cytotoxic T, helper T (Th), regulatory T, and exhausted T cells], with ORs ranging from 1.11 to 1.42. For every 6 months of breastfeeding, we observed increased odds of activated Th-cell infiltration (CD3+CD4+CD69+; OR, 1.13, 95% CI, 0.99–1.29), with a similar association for high-grade serous tumors, but lower odds in clear-cell tumors (OR, 0.43, 95% CI, 0.21–0.87). Conclusions: History of breastfeeding may alter the ovarian tumor immune microenvironment by modulating the abundance of tumor-infiltrating T cells. Impact: Although replication is required, history of breastfeeding may play a role in the activation of the ovarian tumor immune response.