Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer.

BACKGROUND: Obesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion. METHODS: We hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. We tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors. RESULTS: In both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (i.e., BDNF, TNFα, FGF-2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1 in mammary adipose, and reduced phosphorylated FGFR indicating reduced FGF signaling in tumors. CONCLUSIONS: Together, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multi-factorial, and that weight maintenance during the peri-/postmenopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

Diet-Driven Inflammation and Insulinemia and Risk of Interval Breast Cancer.

Interval breast cancers (IBCs) emerge after a non-suspicious mammogram and before the patient's next scheduled screen. Risk factors associated with IBC have not been identified. This study evaluated if the empirical dietary inflammatory pattern (EDIP) or empirical dietary index for hyperinsulinemia (EDIH) scores are associated with IBC compared to screen-detected breast cancer. Data were from women 50-79 years-old in the Women's Health Initiative cohort who completed food frequency questionnaires at baseline (1993-98) and were followed through March 31, 2019 for breast cancer detection. Women were identified as having either IBC diagnosed within 1-year after their last negative screening mammogram (N = 317) or screen-detected breast cancer (N = 1,928). Multivariable-adjusted logistic regression analyses were used to estimate odds ratios for risk of IBC compared to screen-detected cancer in dietary index tertiles. No associations were observed between EDIP or EDIH and IBC. Odds ratios comparing the highest to the lowest dietary index tertile were 1.08; 95%CI, 0.78-1.48 for EDIP and 0.92; 95%CI, 0.67-1.27 for EDIH. The null associations persisted when stratified by BMI categories. Findings suggest that diet-driven inflammation or insulinemia may not be substantially associated with IBC risk among postmenopausal women. Future studies are warranted to identify modifiable factors for IBC prevention.

Erythrocyte membrane fatty acids and breast cancer risk by tumor tissue expression of immuno-inflammatory markers and fatty acid synthase: a nested case-control study.

BACKGROUND: Previous studies of fatty acids and breast cancer risk have shown mixed results, which may be due in part to tumor heterogeneity. Prior research has also illustrated an important role of specific fatty acids in immune regulation, T cell function, and inflammation, indicating that the effects of specific fatty acids on breast cancer risk may vary by tumor expression of immuno-inflammatory markers. We therefore aimed to evaluate the relationships between prediagnostic erythrocyte membrane fatty acids and breast cancer risk by tumor tissue expression of immuno-inflammatory markers (CD4, CD8, CD20, CD163, COX-2) and fatty acid synthase (FAS). METHODS: We conducted a matched case-control study nested within the Nurses' Health Study II (n = 235 cases and 235 controls). Blood samples were collected from 1996 to 1999. Tumor tissue blocks were collected for cases diagnosed after blood collection and through 2006. Unconditional nominal polytomous logistic regression adjusted for matching factors and potential confounders was used to assess whether associations between fatty acids and breast cancer risk varied by tumor expression subtype, ascertained via immunohistochemistry. Odds ratios (OR) and 95% confidence intervals (CI) were estimated separately by tumor expression subtype using unconditional logistic regression. RESULTS: Associations between fatty acids and breast cancer risk did not vary substantially by tumor CD4, CD20, CD163, or COX-2. However, n-3 polyunsaturated fatty acids (PUFAs) were inversely associated with CD8low but not CD8high cancers (CD8low ORT3 vs T1 = 0.45, 95% CI 0.23-0.87, Ptrend = 0.02; CD8high ORT3 vs T1 = 1.19, 95% CI 0.62-2.26, Ptrend = 0.62; Phet = 0.04). n-6 PUFAs were suggestively inversely associated with CD8high but not CD8low cancers (CD8high ORT3 vs T1 = 0.61, 95% CI 0.32-1.14, Ptrend = 0.11; CD8low ORT3 vs T1 = 1.63, 95% CI 0.87-3.04, Ptrend = 0.12; Phet = 0.02). Trans fatty acids were positively associated with FAShigh but not FASlow tumors (FAShigh ORT3 vs T1 = 2.94, 95% CI 1.46-5.91, Ptrend = 0.002; FASlow ORT3 vs T1 = 0.99, 95% CI 0.52-1.92, Ptrend = 0.97; Phet = 0.01). CONCLUSION: Results indicate that the effects of n-3 PUFAs, n-6 PUFAs, and trans fatty acids on breast cancer risk may vary by tumor tissue expression subtypes. Findings suggest potential immuno-modulatory and FAS-mediated mechanisms.

Loss of myoepithelial calponin-1 characterizes high-risk ductal carcinoma in situ cases, which are further stratified by T cell composition.

A hallmark of ductal carcinoma in situ (DCIS) progression is a loss of the surrounding ductal myoepithelium. However, whether compromise in myoepithelial differentiation, rather than overt cellular loss, can be used to predict the risk of DCIS progression is unknown. Here we address this question utilizing pure and mixed DCIS cases (N = 30) as surrogates for DCIS at low and high risk for progression, respectively. We used multiplex immunohistochemical staining to evaluate the relationship between myoepithelial cell differentiation and lymphoid immune cell types associated with poor prognostic DCIS. Our results show that myoepithelial calponin-1 discriminates between pure and mixed DCIS lesions better than histological subtype, presence of necrosis, or nuclear grade. Additionally, focal loss of myoepithelial cells associated with increased PD-1+CD8+ T cells, which suggests a link between the myoepithelium and immune surveillance. To identify associations between calponin-1 expression and immune response, we performed unsupervised hierarchical clustering of myoepithelial and immune cell biomarkers on 219 DCIS lesions from 30 cases. Notably, the majority of pure (low-risk) DCIS lesions clustered in a high calponin-1, T cell low group, whereas the majority of mixed (high-risk) DCIS lesions clustered in a low calponin-1, T cell high group, specifically with CD8+ and PD-1+CD8+ T cells. However, a subset of pure DCIS lesions had a similar calponin-1 and immune signature as the majority of mixed DCIS lesions, which have low calponin-1 and T cell enrichment-raising the possibility that these pure DCIS lesions might be at a high risk for progression.

Metformin inhibits stromal aromatase expression and tumor progression in a rodent model of postmenopausal breast cancer.

BACKGROUND: Obesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases. METHODS: Here, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study. RESULTS: Metformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism. CONCLUSIONS: These findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.

Myoepithelial cell differentiation markers in ductal carcinoma in situ progression.

We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease.

Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.

Women diagnosed with breast cancer within 5 years postpartum have poor survival rates. The process of postpartum mammary gland involution, whereby the lactating gland remodels to its prepregnant state, promotes breast cancer progression in xenograft models. Macrophage influx occurs during mammary gland involution, implicating immune modulation in the promotion of postpartum breast cancer. Herein, we characterize the postpartum murine mammary gland and find an orchestrated influx of immune cells similar to that which occurs during wound healing. Further, the normal involuting gland may be in an immunosuppressed state as discerned by the transient presence of Foxp3(+) regulatory T cells and IL-10(+) macrophages with T cell suppressive function. To determine the influence of the postpartum immune microenvironment on mammary tumor promotion, we developed an immune-competent model. In this model, mammary tumors in the involution group are sixfold larger than nulliparous group tumors, have decreased CD4(+) and CD8(+) T cell infiltrates and contain a greater number of macrophages with the ability to inhibit T cell activation. Targeting involution with a neutralizing antibody against the immunosuppressive cytokine IL-10 reduces tumor growth in involution group mice but not in nulliparous mice, implicating the involution microenvironment as the primary target of αIL-10 treatment. Relevance to women is implicated, as we find postlactational human breast tissue has transient high IL-10(+) and Foxp3(+) immune cell infiltrate. These data show an immune modulated microenvironment within the normal involuting mammary gland suggestive of immunosuppression, that when targeted reduces tumor promotion, revealing possible immune-based strategies for postpartum breast cancer.

Physiological COX-2 expression in breast epithelium associates with COX-2 levels in ductal carcinoma in situ and invasive breast cancer in young women.

Cyclooxygenase-2 (COX-2) overexpression is implicated in increased risk and poorer outcomes in breast cancer in young women. We investigated COX-2 regulation in normal premenopausal breast tissue and its relationship to malignancy in young women. Quantitative COX-2 immunohistochemistry was performed on adjacent normal and breast cancer tissues from 96 premenopausal women with known clinical reproductive histories, and on rat mammary glands with distinct ovarian hormone exposures. COX-2 expression in the normal breast epithelium varied more than 40-fold between women and was associated with COX-2 expression levels in ductal carcinoma in situ and invasive cancer. Normal breast COX-2 expression was independent of known breast cancer prognostic indicators, including tumor stage and clinical subtype, indicating that factors regulating physiological COX-2 expression may be the primary drivers of COX-2 expression in breast cancer. Ovarian hormones, particularly at pregnancy levels, were identified as modulators of COX-2 in normal mammary epithelium. However, serial breast biopsy analysis in nonpregnant premenopausal women suggested relatively stable baseline levels of COX-2 expression, which persisted independent of menstrual cycling. These data provide impetus to investigate how baseline COX-2 expression is regulated in premenopausal breast tissue because COX-2 levels in normal breast epithelium may prove to be an indicator of breast cancer risk in young women, and predict the chemopreventive and therapeutic efficacy of COX-2 inhibitors in this population.

Mammary gland involution as an immunotherapeutic target for postpartum breast cancer.

Postpartum mammary gland involution has been identified as tumor-promotional and is proposed to contribute to the increased rates of metastasis and poor survival observed in postpartum breast cancer patients. In rodent models, the involuting mammary gland microenvironment is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Postpartum involution shares many attributes with wound healing, including upregulation of genes involved in immune responsiveness and infiltration of tissue by immune cells. In rodent models, treatment with non-steroidal anti-inflammatory drugs (NSAIDs) ameliorates the tumor-promotional effects of involution, consistent with the immune milieu of the involuting gland contributing to tumor promotion. Currently, immunotherapy is being investigated as a means of breast cancer treatment with the purpose of identifying ways to enhance anti-tumor immune responses. Here we review evidence for postpartum mammary gland involution being a uniquely defined 'hot-spot' of pro-tumorigenic immune cell infiltration, and propose that immunotherapy should be explored for prevention and treatment of breast cancers that arise in this environment.

Postpartum breast involution reveals regression of secretory lobules mediated by tissue-remodeling.

INTRODUCTION: A postpartum diagnosis of breast cancer is an independent predictor of metastases, however the reason is unknown. In rodents, the window of postpartum mammary gland involution promotes tumor progression, suggesting a role for breast involution in the poor prognosis of human postpartum breast cancers. Rodent mammary gland involution is characterized by the programmed elimination of the secretory lobules laid down in preparation for lactation. This tissue involution process involves massive epithelial cell death, stromal remodeling, and immune cell infiltration with similarities to microenvironments present during wound healing and tumor progression. Here, we characterize breast tissue from premenopausal women with known reproductive histories to determine the extent, duration and cellular mechanisms of postpartum lobular involution in women. METHODS: Adjacent normal breast tissues from premenopausal women (n = 183) aged 20 to 45 years, grouped by reproductive categories of nulliparous, pregnant and lactating, and by time since last delivery were evaluated histologically and by special stain for lobular area, lobular type composition, apoptosis and immune cell infiltration using computer assisted quantitative methods. RESULTS: Human nulliparous glands were composed dominantly of small (approximately 10 acini per lobule) and medium (approximately 35 acini per lobule) sized lobules. With pregnancy and lactation, a >10 fold increase in breast epithelial area was observed compared to nulliparous cases, and lactating glands were dominated by mature lobules (>100 acini per lobule) with secretory morphology. Significant losses in mammary epithelial area and mature lobule phenotypes were observed within 12 months postpartum. By 18 months postpartum, lobular area content and lobule composition were indistinguishable from nulliparous cases, data consistent with postpartum involution facilitating regression of the secretory lobules developed in preparation for lactation. Analyses of apoptosis and immune cell infiltrate confirmed that human postpartum breast involution is characterized by wound healing-like tissue remodeling programs that occur within a narrowed time frame. CONCLUSIONS: Human postpartum breast involution is a dominant tissue-remodeling process that returns the total lobular area of the gland to a level essentially indistinguishable from the nulliparous gland. Further research is warranted to determine whether the normal physiologic process of postpartum involution contributes to the poor prognosis of postpartum breast cancer.

TSG-6+ cancer-associated fibroblasts modulate myeloid cell responses and impair anti-tumor response to immune checkpoint therapy in pancreatic cancer.

Resistance to immune checkpoint therapy (ICT) presents a growing clinical challenge. The tumor microenvironment (TME) and its components, namely tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), play a pivotal role in ICT resistance; however, the underlying mechanisms remain under investigation. In this study, we identify expression of TNF-Stimulated Factor 6 (TSG-6) in ICT-resistant pancreatic tumors, compared to ICT-sensitive melanoma tumors, both in mouse and human. TSG-6 is expressed by CAFs within the TME, where suppressive macrophages expressing Arg1, Mafb, and Mrc1, along with TSG-6 ligand Cd44, predominate. Furthermore, TSG-6 expressing CAFs co-localize with the CD44 expressing macrophages in the TME. TSG-6 inhibition in combination with ICT improves therapy response and survival in pancreatic tumor-bearing mice by reducing macrophages expressing immunosuppressive phenotypes and increasing CD8 T cells. Overall, our findings propose TSG-6 as a therapeutic target to enhance ICT response in non-responsive tumors.

Durvalumab and tremelimumab before surgery in patients with hormone receptor positive, HER2-negative stage II-III breast cancer.

A clinical trial was conducted to assess the feasibility of enrolling patients with Stage II or III hormone receptor positive (HR+)/HER2-negative breast cancer to pre-operative dual PD-L1/CTLA-4 checkpoint inhibition administered prior to neoadjuvant chemotherapy (NACT). Eight eligible patients were treated with upfront durvalumab and tremelimumab for two cycles. Patients then received NACT prior to breast surgery. Seven patients had baseline and interval breast ultrasounds after combination immunotherapy and the responses were mixed: 3/7 patients experienced a ≥30% decrease in tumor volume, 3/7 a ≥30% increase, and 1 patient had stable disease. At the time of breast surgery, 1/8 patients had a pathologic complete response (pCR). The trial was stopped early after 3 of 8 patients experienced immunotherapy-related toxicity or suspected disease progression that prompted discontinuation or a delay in the administration of NACT. Two patients experienced grade 3 immune-related adverse events (1 with colitis, 1 with endocrinopathy). Analysis of the tumor microenvironment after combination immunotherapy did not show a significant change in immune cell subsets from baseline. There was limited benefit for dual checkpoint blockade administered prior to NACT in our study of 8 patients with HR+/HER2-negative breast cancer.

A Modular Trial of Androgen Signaling Inhibitor Combinations Testing a Risk-Adapted Strategy in Patients with Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To determine the efficacy and safety of risk-adapted combinations of androgen signaling inhibitors and inform disease classifiers for metastatic castration-resistant prostate cancers. PATIENTS AND METHODS: In a modular, randomized phase II trial, 192 men were treated with 8 weeks of abiraterone acetate, prednisone, and apalutamide (AAPA; module 1) and then allocated to modules 2 or 3 based on satisfactory (≥50% PSA decline from baseline and <5 circulating tumor cell/7.5 mL) versus unsatisfactory status. Men in the former were randomly assigned to continue AAPA alone (module 2A) or with ipilimumab (module 2B). Men in the latter group had carboplatin + cabazitaxel added to AAPA (module 3). Optional baseline biopsies were subjected to correlative studies. RESULTS: Median overall survival (from allocation) was 46.4 [95% confidence interval (CI), 39.2-68.2], 41.4 (95% CI, 33.3-49.9), and 18.7 (95% CI, 14.3-26.3) months in modules 2A (n = 64), 2B (n = 64), and 3 (n = 59), respectively. Toxicities were within expectations. Of 192 eligible patients, 154 (80.2%) underwent pretreatment metastatic biopsies. The aggressive-variant prostate cancer molecular profile (defects in ≥2 of p53, RB1, and PTEN) was associated with unsatisfactory status. Exploratory analyses suggested that secreted phosphoprotein 1-positive and insulin-like growth factor-binding protein 2-positive macrophages, druggable myeloid cell markers, and germline pathogenic mutations were enriched in the unsatisfactory group. CONCLUSIONS: Adding ipilimumab to AAPA did not improve outcomes in men with androgen-responsive metastatic castration-resistant prostate cancer. Despite the addition of carboplatin + cabazitaxel, men in the unsatisfactory group had shortened survivals. Adaptive designs can enrich for biologically and clinically relevant disease subgroups to contribute to the development of marker-informed, risk-adapted therapy strategies in men with prostate cancer.

Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer.

Previous studies report conflicting data on outcomes of pregnancy-associated breast cancer (PABC). Our aim was to examine the effect of a postpartum diagnosis on maternal prognosis in a young women's breast cancer cohort. We conducted a retrospective cohort study of women age ≤45 years, diagnosed with breast cancer (n = 619) during 1981-2011 at the University of Colorado Hospital and The Shaw Cancer Center in Edwards, CO. Breast cancer cases were grouped according to time between giving birth and diagnosis: nulliparous (n = 125), pregnant (n = 24), < 5 years postpartum (n = 136), >5-<10 postpartum (n = 130), and ≥10 years postpartum (n = 147), to examine the clinicopathologic features and the risk of distance recurrence and death. Cases diagnosed after pregnancy, but within five-years postpartum, had an approximate three fold increased risk of distant recurrence (HR 2.80, 95 % CI: 1.12-6.57) and death (HR 2.65, 95 % CI: 1.09-6.42) compared to nulliparous cases. Postpartum cases diagnosed within five years of last childbirth demonstrated a higher five-year distant recurrence probability (31.1 %) and a markedly lower five-year overall survival probability (65.8 %) compared to nulliparous cases (14.8 and 98.0 %, respectively). A diagnosis of breast cancer during the first five-years postpartum confers poorer maternal prognoses after adjustment for biologic subtype, stage, and year of diagnosis. We propose that the definition of PABC should include cases diagnosed up to at least five-years postpartum to better delineate the increased risk imparted by a postpartum diagnosis. Based on emerging preclinical and epidemiologic data, we propose that pregnant and postpartum cases be researched as distinct subsets of PABC to clarify the risk imparted by pregnancy and the events subsequent to pregnancy, such as breast involution, on breast cancer. Further, we highlight the importance of postpartum breast cancer as an area for further research to reduce the increased metastatic potential and mortality of PABC.

Myeloid-specific KDM6B inhibition sensitizes glioblastoma to PD1 blockade.

Glioblastoma (GBM) tumors are enriched in immune-suppressive myeloid cells and are refractory to immune checkpoint therapy (ICT). Targeting epigenetic pathways to reprogram the functional phenotype of immune-suppressive myeloid cells to overcome resistance to ICT remains unexplored. Single-cell and spatial transcriptomic analyses of human GBM tumors demonstrated high expression of an epigenetic enzyme-histone 3 lysine 27 demethylase (KDM6B)-in intratumoral immune-suppressive myeloid cell subsets. Importantly, myeloid cell-specific Kdm6b deletion enhanced proinflammatory pathways and improved survival in GBM tumor-bearing mice. Mechanistic studies showed that the absence of Kdm6b enhances antigen presentation, interferon response and phagocytosis in myeloid cells by inhibition of mediators of immune suppression including Mafb, Socs3 and Sirpa. Further, pharmacological inhibition of KDM6B mirrored the functional phenotype of Kdm6b-deleted myeloid cells and enhanced anti-PD1 efficacy. This study thus identified KDM6B as an epigenetic regulator of the functional phenotype of myeloid cell subsets and a potential therapeutic target for enhanced response to ICT.

Immune and pathologic responses in patients with localized prostate cancer who received daratumumab (anti-CD38) or edicotinib (CSF-1R inhibitor).

BACKGROUND: The prostate tumor microenvironment (TME) is immunosuppressive, with few effector T cells and enrichment of inhibitory immune populations, leading to limited responses to treatments such as immune checkpoint therapies (ICTs). The immune composition of the prostate TME differs across soft tissue and bone, the most common site of treatment-refractory metastasis. Understanding immunosuppressive mechanisms specific to prostate TMEs will enable rational immunotherapy strategies to generate effective antitumor immune responses. Daratumumab (anti-CD38 antibody) and edicotinib (colony-stimulating factor-1 receptor (CSF-1R) inhibitor) may alter the balance within the prostate TME to promote antitumor immune responses. HYPOTHESIS: Daratumumab or edicotinib will be safe and will alter the immune TME, leading to antitumor responses in localized prostate cancer. PATIENTS AND METHODS: In this presurgical study, patients with localized prostate cancer received 4 weekly doses of daratumumab or 4 weeks of daily edicotinib prior to radical prostatectomy (RP). Treated and untreated control (Gleason score ≥8 in prostate biopsy) prostatectomy specimens and patient-matched pre- and post-treatment peripheral blood mononuclear cells (PBMCs) and bone marrow samples were evaluated. The primary endpoint was incidence of adverse events (AEs). The secondary endpoint was pathologic complete remission (pCR) rate. RESULTS: Twenty-five patients were treated (daratumumab, n=15; edicotinib, n=10). All patients underwent RP without delays. Grade 3 treatment-related AEs with daratumumab occurred in 3 patients (12%), and no ≥grade 3 treatment-related AEs occurred with edicotinib. No changes in serum prostate-specific antigen (PSA) levels or pCRs were observed. Daratumumab led to a decreased frequency of CD38+ T cells, natural killer cells, and myeloid cells in prostate tumors, bone marrow, and PBMCs. There were no consistent changes in CSF-1R+ immune cells in prostate, bone marrow, or PBMCs with edicotinib. Neither treatment induced T cell infiltration into the prostate TME. CONCLUSIONS: Daratumumab and edicotinib treatment was safe and well-tolerated in patients with localized prostate cancer but did not induce pCRs. Decreases in CD38+ immune cells were observed in prostate tumors, bone marrow, and PBMCs with daratumumab, but changes in CSF-1R+ immune cells were not consistently observed with edicotinib. Neither myeloid-targeted agent alone was sufficient to generate antitumor responses in prostate cancer; thus, combinations with agents to induce T cell infiltration (eg, ICTs) will be needed to overcome the immunosuppressive prostate TME.

Inflammation induced by tumor-associated nerves promotes resistance to anti-PD-1 therapy in cancer patients and is targetable by interleukin-6 blockade.

While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.

Exercise Training Reduces the Inflammatory Response and Promotes Intestinal Mucosa-Associated Immunity in Lynch Syndrome.

PURPOSE: Lynch syndrome (LS) is a hereditary condition with a high lifetime risk of colorectal and endometrial cancers. Exercise is a non-pharmacologic intervention to reduce cancer risk, though its impact on patients with LS has not been prospectively studied. Here, we evaluated the impact of a 12-month aerobic exercise cycling intervention in the biology of the immune system in LS carriers. PATIENTS AND METHODS: To address this, we enrolled 21 patients with LS onto a non-randomized, sequential intervention assignation, clinical trial to assess the effect of a 12-month exercise program that included cycling classes 3 times weekly for 45 minutes versus usual care with a one-time exercise counseling session as control. We analyzed the effects of exercise on cardiorespiratory fitness, circulating, and colorectal-tissue biomarkers using metabolomics, gene expression by bulk mRNA sequencing, and spatial transcriptomics by NanoString GeoMx. RESULTS: We observed a significant increase in oxygen consumption (VO2peak) as a primary outcome of the exercise and a decrease in inflammatory markers (prostaglandin E) in colon and blood as the secondary outcomes in the exercise versus usual care group. Gene expression profiling and spatial transcriptomics on available colon biopsies revealed an increase in the colonic mucosa levels of natural killer and CD8+ T cells in the exercise group that were further confirmed by IHC studies. CONCLUSIONS: Together these data have important implications for cancer interception in LS, and document for the first-time biological effects of exercise in the immune system of a target organ in patients at-risk for cancer.

Investigative needle core biopsies for multi-omics in Glioblastoma.

Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.

Young-Onset Breast Cancer Outcomes by Time Since Recent Childbirth in Utah.

Importance: Breast cancer diagnosed within 5 to 10 years after childbirth, called postpartum breast cancer (PPBC), is associated with increased risk for metastasis and death. Whether a postpartum diagnosis is an independent risk factor or a surrogate marker of cancer features associated with poor outcomes remains understudied. Objective: To determine whether diagnostic temporal proximity to childbirth is associated with features of breast cancer associated with poor outcomes, including tumor stage, estrogen receptor (ER) status, and risk for distant metastasis and breast cancer-specific mortality, using a population database from the state of Utah. Design, Setting, and Participants: This population-based cohort study using the Utah Population Database (UPDB) included individuals with stage I to III breast cancer diagnosed at age 45 years or younger between 1996 and 2017, followed-up until February 2020. Participant data were analyzed from November 2019 to August 2022. Exposure: The primary exposures were no prior childbirth or time between most recent childbirth and breast cancer diagnosis. Patients were grouped by diagnoses within less than 5 years, 5 to less than 10 years, or 10 years or more since recent childbirth. Main Outcomes and Measures: The 2 primary outcomes were distant metastasis-free survival and breast cancer-specific death. Cox proportional hazard models were used to investigate associations between exposures and outcomes adjusting for diagnosis year, patient age, tumor stage, and estrogen receptor (ER) status. Results: Of 2970 individuals with breast cancer diagnosed at age 45 years or younger (mean [SD] age, 39.3 [5.0] years; 12 Black individuals [0.4%], 2679 White individuals [90.2%]), breast cancer diagnosis within 5 years of recent childbirth was independently associated with approximately 1.5-fold elevated risk for metastasis (hazard ratio [HR], 1.5; 95% CI, 1.2-2.0) and breast cancer-specific death (HR, 1.5; 95% CI, 1.1-2.1) compared with nulliparous individuals. For cancers classically considered to have tumor features associated with good outcomes (ie, stage I or II and ER-positive), a postpartum diagnosis was a dominant feature associated with increased risk for metastasis and death (eg, for individuals with ER-positive disease diagnosed within <5 years of childbirth: age-adjusted metastasis HR, 1.5; 95% CI, 1.1-2.1; P = .01; age-adjusted death HR, 1.5; 95% CI, 1.0-2.1; P = .04) compared with nulliparous individuals. Furthermore, liver metastases were specifically increased in the group with diagnosis within 5 years postpartum and with positive ER expression (38 of 83 patients [45.8%]) compared with the nulliparous (28 of 77 patients [36.4%]), although the difference was not statistically significant. Overall, these data implicate parity-associated breast and liver biology in the observed poor outcomes of PPBC. Conclusions and Relevance: In this cohort study of individuals with breast cancer diagnosed at age 45 years or younger, a postpartum breast cancer diagnosis was a risk factor associated with poor outcomes. Irrespective of ER status, clinical consideration of time between most recent childbirth and breast cancer diagnosis could increase accuracy of prognosis in patients with young-onset breast cancer.