An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer.

OBJECTIVES: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

Racial disparities in disease-specific mortality and surgical management of patients with ductal carcinoma in situ with microinvasion.

BACKGROUND AND OBJECTIVES: Given persistent racial disparities in breast cancer outcomes, this study explores racial differences in disease-specific mortality and surgical management among patients with microinvasive ductal carcinoma in situ (DCIS-MI). METHODS: The Surveillance, Epidemiology, and End Results Program was queried for patients aged 18+ years with DCIS-MI between January 1, 2010 and December 31, 2018. The study cohort was divided into non-Hispanic Black (NHB) and non-Hispanic White (NHW) patients. Disease-specific mortality was evaluated using Cox proportional hazards models. RESULTS: A total of 3400 patients were identified, of which 569 (16.7%) were NHB and 2831 (83.3%) were NHW. Compared with NHW patients, NHB patients had more positive lymph nodes (7.6% vs. 3.9% p < 0.001). In addition, NHB women were more likely to undergo axillary lymph node dissection (6.0% vs. 3.8%, p = 0.044) and receive chemotherapy (11.8% vs. 7.2%, p < 0.001). There were no racial differences in breast surgery type (p = 0.168), reconstructive surgery (p = 0.362), or radiation therapy (p = 0.342). Overall, NHB patients had worse disease-specific mortality (adjusted hazard ratio 2.13, 95% confidence interval [CI]: 1.10-4.14) with mortality risks diverging from NHW women after 3 years (6 years rate ratio [RR] 2.12, 95% CI: 1.13-4.34; 9 years RR 2.32, 95% CI: 1.24-4.35). CONCLUSIONS: NHB women with DCIS-MI present with higher nodal disease burden and experience worse disease-specific mortality than NHW women.

Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

Association Between Neighborhood Opportunity, Allostatic Load, and All-Cause Mortality in Patients With Breast Cancer.

PURPOSE: Adverse neighborhood contextual factors may affect breast cancer outcomes through environmental, psychosocial, and biological pathways. The objective of this study is to examine the relationship between allostatic load (AL), neighborhood opportunity, and all-cause mortality among patients with breast cancer. METHODS: Women age 18 years and older with newly diagnosed stage I-III breast cancer who received surgical treatment between January 1, 2012, and December 31, 2020, at a National Cancer Institute Comprehensive Cancer Center were identified. Neighborhood opportunity was operationalized using the 2014-2018 Ohio Opportunity Index (OOI), a composite measure derived from neighborhood level transportation, education, employment, health, housing, crime, and environment. Logistic and Cox regression models tested associations between the OOI, AL, and all-cause mortality. RESULTS: The study cohort included 4,089 patients. Residence in neighborhoods with low OOI was associated with high AL (adjusted odds ratio, 1.21 [95% CI, 1.05 to 1.40]). On adjusted analysis, low OOI was associated with greater risk of all-cause mortality (adjusted hazard ratio [aHR], 1.45 [95% CI, 1.11 to 1.89]). Relative to the highest (99th percentile) level of opportunity, risk of all-cause mortality steeply increased up to the 70th percentile, at which point the rate of increase plateaued. There was no interaction between the composite OOI and AL on all-cause mortality (P = .12). However, there was a higher mortality risk among patients with high AL residing in lower-opportunity environments (aHR, 1.96), but not in higher-opportunity environments (aHR, 1.02; P interaction = .02). CONCLUSION: Lower neighborhood opportunity was associated with higher AL and greater risk of all-cause mortality among patients with breast cancer. Additionally, environmental factors and AL interacted to influence all-cause mortality. Future studies should focus on interventions at the neighborhood and individual level to address socioeconomically based disparities in breast cancer.

Targeting metabolic sensing switch GPR84 on macrophages for cancer immunotherapy.

INTRODUCTION: As one of the major components of the tumor microenvironment, tumor-associated macrophages (TAMs) possess profound inhibitory activity against T cells and facilitate tumor escape from immune checkpoint blockade therapy. Converting this pro-tumorigenic toward the anti-tumorigenic phenotype thus is an important strategy for enhancing adaptive immunity against cancer. However, a plethora of mechanisms have been described for pro-tumorigenic differentiation in cancer, metabolic switches to program the anti-tumorigenic property of TAMs are elusive. MATERIALS AND METHODS: From an unbiased analysis of single-cell transcriptome data from multiple tumor models, we discovered that anti-tumorigenic TAMs uniquely express elevated levels of a specific fatty acid receptor, G-protein-coupled receptor 84 (GPR84). Genetic ablation of GPR84 in mice leads to impaired pro-inflammatory polarization of macrophages, while enhancing their anti-inflammatory phenotype. By contrast, GPR84 activation by its agonist, 6-n-octylaminouracil (6-OAU), potentiates pro-inflammatory phenotype via the enhanced STAT1 pathway. Moreover, 6-OAU treatment significantly retards tumor growth and increases the anti-tumor efficacy of anti-PD-1 therapy. CONCLUSION: Overall, we report a previously unappreciated fatty acid receptor, GPR84, that serves as an important metabolic sensing switch for orchestrating anti-tumorigenic macrophage polarization. Pharmacological agonists of GPR84 hold promise to reshape and reverse the immunosuppressive TME, and thereby restore responsiveness of cancer to overcome resistance to immune checkpoint blockade.

Roles of Androgen Receptor Signaling in Urothelial Carcinoma.

Preclinical and clinical data suggest that androgen receptor signaling strongly contributes to bladder cancer development. The roles of the androgen receptor in bladder carcinogenesis have obvious implications for understanding the strong male sex bias in this disease and for potential therapeutic strategies as well. In this review, we summarize what is known about androgen receptor signaling in urothelial carcinoma as well as in tumor-infiltrating immune cells, reviewing preclinical and clinical data. We also highlight clinical trial efforts in this area.

Single-Cell RNA-Seq Analysis of Patient Myeloid-Derived Suppressor Cells and the Response to Inhibition of Bruton's Tyrosine Kinase.

Myeloid-derived suppressor cell (MDSC) levels are elevated in patients with cancer and contribute to reduced efficacy of immune checkpoint therapy. MDSC express Bruton's tyrosine kinase (BTK) and BTK inhibition with ibrutinib, an FDA-approved irreversible inhibitor of BTK, leads to reduced MDSC expansion/function in mice and significantly improves the antitumor activity of anti-PD-1 antibody treatments. Single-cell RNA sequencing (scRNA-seq) was used to characterize the effect of ibrutinib on gene expression of fluorescence-activated cell sorting-enriched MDSC from patients with different cancer types [breast, melanoma, head and neck squamous cell cancer (HNSCC)]. Melanoma patient MDSC were treated in vitro for 4 hours with 5 µmol/L ibrutinib or DMSO, processed for scRNA-seq using the Chromium 10× Genomics platform, and analyzed via the Seurat v4 standard integrative workflow. Baseline gene expression of MDSC from patients with breast, melanoma, and HNSCC cancer revealed similarities among the top expressed genes. In vitro ibrutinib treatment of MDSC from patients with melanoma resulted in significant changes in gene expression. GBP1, IL-1ß, and CXCL8 were among the top downregulated genes whereas RGS2 and ABHD5 were among the top upregulated genes (P < 0.001). Double positive CD14+CD15+ MDSC and PMN-MDSC responded similarly to BTK inhibition and exhibited more pronounced gene changes compared with early MDSC and M-MDSC. Pathway analysis revealed significantly downregulated pathways including TREM1, nitric oxide signaling, and IL-6 signaling (P < 0.004). IMPLICATIONS: scRNA-seq revealed characteristic gene expression patterns for MDSC from different patients with cancer and BTK inhibition led to the downregulation of multiple genes and pathways important to MDSC function and migration.

Racial Disparities in Breast Cancer: from Detection to Treatment.

PURPOSE OF REVIEW: Update on current racial disparities in the detection and treatment of breast cancer. RECENT FINDINGS: Breast cancer remains the leading cause of cancer death among Black and Hispanic women. Mammography rates among Black and Hispanic women have surpassed those among White women, with studies now advocating for earlier initiation of breast cancer screening in Black women. Black, Hispanic, Asian, and American Indian and Alaskan Native women continue to experience delays in diagnosis and time to treatment. Further, racial discrepancies in receipt of guideline-concordant care, access to genetic testing and surgical reconstruction persist. Disparities in the initiation, completion, toxicity, and efficacy of chemotherapy, endocrine therapy, and targeted drug therapy remain for racially marginalized women. Efforts to evaluate the impact of race and ethnicity across the breast cancer spectrum are increasing, but knowledge gaps remain and further research is necessary to reduce the disparity gap.

The Implications of Racialized Economic Segregation and Allostatic Load on Mortality in Patients with Breast Cancer.

BACKGROUND: The objective of this study was to examine the association between racialized economic segregation, allostatic load (AL), and all-cause mortality in patients with breast cancer. PATIENTS AND METHODS: Women aged 18+ years with stage I-III breast cancer diagnosed between 01/01/2012 and 31/12/2020 were identified in the Ohio State University cancer registry. Racialized economic segregation was measured at the census tract level using the index of concentration at the extremes (ICE). AL was calculated with biomarkers from the cardiac, metabolic, immune, and renal systems. High AL was defined as AL greater than the median. Univariable and multivariable regression analyses using restricted cubic splines examined the association between racialized economic segregation, AL, and all-cause mortality. RESULTS: Among 4296 patients, patients residing in neighborhoods with the highest racialized economic segregation (Q1 versus Q4) were more likely to be Black (25% versus 2.1%, p < 0.001) and have triple-negative breast cancer (18.2% versus 11.6%, p < 0.001). High versus low racialized economic segregation was associated with high AL [adjusted odds ratio (aOR) 1.40, 95% confidence interval (CI) 1.21-1.61] and worse all-cause mortality [adjusted hazard ratio (aHR) 1.41, 95% CI 1.08-1.83]. In dose-response analyses, patients in lower segregated neighborhoods (relative to the 95th percentile) had lower odds of high AL, whereas patients in more segregated neighborhoods had a non-linear increase in the odds of high AL. DISCUSSION: Racialized economic segregation is associated with high AL and a greater risk of all-cause mortality in patients with breast cancer. Additional studies are needed to elucidate the causal pathways and mechanisms linking AL, neighborhood factors, and patient outcomes.

Analysis of Changes in Plasma Cytokine Levels in Response to IL12 Therapy in Three Clinical Trials.

The ability of IL12 to stimulate natural killer (NK) cell and T-cell antitumor activity makes it an attractive candidate for the immune therapy of cancer. Our group has demonstrated that IL12 enhances the NK cell response to antibody-coated tumor cells and conducted three clinical trials utilizing IL12 with mAbs (OSU-9968, OSU-0167, and OSU-11010). To better characterize IL12-induced immunity, plasma cytokine levels were measured in 21 patients from these trials with favorable and unfavorable responses. t-statistics and linear modeling were used to test for differences within and between response groups by examining levels at baseline and post-IL12 administration. Patients exhibited significant increases in 11 cytokines post-IL12 administration when analyzed collectively. However, several cytokines were differentially induced by IL12 depending on response. GMCSF was significantly increased in complete/partially responding patients, while stable disease patients had significant increases in IL10 and decreases in VEGF-C. Patients who experienced progressive disease had significant increases in CCL3, CCL4, IL18, TNFα, CXCL10, CCL8, CCL2, IL6, and IFNγ. The increases in CCL3, CCL4, and IL6 in progressive disease patients were significantly higher than in clinically benefitting patients and most prominent within the first two cycles of IL12 therapy. This correlative pilot study has identified changes that occur in levels of circulating cytokines following IL12 administration to patients with cancer, but this report must be viewed as exploratory in nature. It is meant to spark further inquiry into the topic via the analysis of additional cohorts of patients with similar characteristics who have received IL12 in a uniform fashion. SIGNIFICANCE: IL12 activates immune cells and is used to treat cancer. The profile of circulating cytokines was measured in an exploratory fashion in patients with cancer that received IL12 in combination with mAbs. This correlative pilot study could serve as the basis for additional studies of IL12 effects on the production of immune cytokines.

Short-term and long-term financial toxicity from breast cancer treatment: a qualitative study.

PURPOSE: The rising cost of breast cancer treatment has increased patients' financial burden, intensifying an already stressful treatment process. Although researchers increasingly recognize the harmful impact of medical and nonmedical costs associated with cancer treatment, understanding patients' perspectives of financial toxicity is limited. We aimed to explore the topic of financial toxicity through the lived experiences of patients with breast cancer from groups at risk of social and economic marginalization. METHODS: We conducted semi-structured interviews with 50 women with breast cancer from four specific groups: Black women, Medicaid enrollees, rural residents, and women age ≤ 40. We transcribed, coded, and analyzed the data using deductive and inductive approaches. RESULTS: Two overarching themes captured patients' experiences of financial toxicity: short-term and long-term impacts. Short-term stressors included direct medical (e.g., co-pays, premiums), nonmedical (e.g., transportation, lodging), and indirect (e.g., job loss, reduced work hours) costs. Early in their treatments, patients' focus on survival took precedence over financial concerns. However, as the treatment course progressed, fear of consequences from compounding costs of care and financial distress negatively impacted patients' lifestyles and outlooks for the future. CONCLUSION: Programs addressing financial toxicity that look beyond early-phase interventions are needed. Specifically, patients struggling with the accumulation of treatment costs and the resultant stress require ongoing support. Long-term support is especially needed for groups vulnerable to financial instability and social marginalization.

Low Hospital Volume Is Associated with Higher All-Cause Mortality in Black Women with Triple Negative Breast Cancer.

INTRODUCTION: This study examines the association between hospital volume and all-cause mortality in Black women with triple negative breast cancer (TNBC) who received surgery and chemotherapy. METHODS: Black women ages 18+ with stage I-III TNBC who received both surgery and chemotherapy were identified in the National Cancer Database (NCDB). Hospital volume was determined using the number of annual breast cancer cases divided by the number of years the hospital participated in the NCDB. Hospital annual volume quartiles ranged from Q1 (lowest) to Q4 (highest). Univariable analysis and multivariable logistic regression modeling with restricted cubic splines examined the effect of hospital volume on all-cause mortality. RESULTS: Sixteen thousand five hundred fifty-six patients met the study criteria. All-cause mortality incidence was lower at higher volume compared to lower volume hospitals Q1 24.1% (95% CI: 22.8 to 25.4), Q2 21.8% (95% CI: 20.5 to 23.1), Q3 20.9% (95% CI: 19.6 to 22.1), Q4 19.0% (95% CI: 17.7 to 20.1), p<0.001. On multivariable analysis, treatment at the highest hospital volume quartile was associated with a 21% reduction in the odds of death compared to the lowest quartile [Q4 Vs. Q1, OR=0.79 (95% CI: 0.67 to 0.92)]. For every 100-patient increase in annual volume, all-cause mortality was reduced by 4% [OR=0.96 (95% CI: 0.94 to 0.98)]. There was a significant linear dose-dependent relationship between increasing hospital volume and all-cause mortality. CONCLUSION: Black women treated at high-volume hospitals have lower all-cause mortality than those at low-volume hospitals. Future studies should examine the characteristics of high-volume hospitals associated with improved outcomes.

Phase Ib study of HSP90 inhibitor, onalespib (AT13387), in combination with paclitaxel in patients with advanced triple-negative breast cancer.

Background: Heat shock protein 90 (HSP90) is a molecular chaperone required for stabilization of client proteins over-activated in triple-negative breast cancer (TNBC). Over-expression of HSP90 client proteins has been implicated in paclitaxel resistance. Onalespib (AT13387) is a potent inhibitor of HSP90 that could improve paclitaxel efficacy when administered in combination. Design: This phase Ib trial administered onalespib with paclitaxel in patients with advanced TNBC to assess safety and establish a recommended phase II dose (RP2D). Objectives: The primary objectives were determining the dose-limiting toxicities and maximum tolerated dose of combination therapy. Secondary objectives included pharmacokinetic (PK) analysis and determination of overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS). Methods: Patients with advanced TNBC were treated with standard dose intravenous paclitaxel in combination with intravenous onalespib at doses ranging from 120 to 260 mg/m2 administered on days 1, 8, and 15 of a 28-day cycle using a standard 3 + 3 design. A total of 15 patients were enrolled to dose expansion cohort at RP2D to confirm safety profile. Results: Thirty-one patients were enrolled in the study, of which over 90% had received prior taxane therapy. Paclitaxel was given for metastatic disease in 23% of patients. Adverse events (AEs) included anemia (grade 3: 20%), lymphopenia (grade 3: 17%), and neutropenia (grade 3: 33%, grade 4: 4%). The most frequent grade ⩾3 non-hematologic AE was diarrhea (7%). The established RP2D was 260 mg/m2 onalespib when given with paclitaxel at 80 mg/m2. PK analysis revealed a modest drug interaction profile for onalespib in the combination regimen. ORR was 20%. Three patients achieved complete responses, all of whom had received prior taxane therapy. Median DOR was 5.6 months; median PFS was 2.9 months. Conclusion: Combination treatment with onalespib and paclitaxel had an acceptable toxicity profile and RP2D was determined to be 260 mg/m2 of onalespib. Combination therapy showed antitumor activity in patients with advanced TNBC. Trial registration: Onalespib and paclitaxel in treating patients with advanced TNBC https://clinicaltrials.gov/ct2/show/NCT02474173.

Phase 1b study of HSP90 inhibitor called onalespib in combination with paclitaxel in patients with advanced triple-negative breast cancer This Phase 1b study demonstrated that treatment with a combination of onalespib and paclitaxel was reasonably well tolerated by most patients. Onalespib at 260 mg/m² given intravenously on days 1, 8 and 15 on 28-day cycles in combination with standard dose and schedule of paclitaxel was established as the recommended phase 2 dose for further clinical development. Despite minor drug-drug interactions between these 2 agents, onalespib did not alter paclitaxel exposure and paclitaxel did not affect exposure to onalespib. While onalespib with paclitaxel combination therapy did not yield durable objective responses or prolonged progression-free survival, there were several patients with long-lasting benefit from this combination including patients who previously experienced progression on taxane therapy.

Depression, Inflammation, and Intestinal Permeability: Associations with Subjective and Objective Cognitive Functioning throughout Breast Cancer Survivorship.

About one-in-three breast cancer survivors have lingering cognitive complaints and objective cognitive impairment. Chronic inflammation and intestinal permeability (i.e., leaky gut), two risk factors for cognitive decline, can also fuel depression-another vulnerability for cognitive decline. The current study tested whether depression accompanied by high levels of inflammation or intestinal permeability predicted lower subjective and objective cognitive function in breast cancer survivors. We combined data from four breast cancer survivor studies (n = 613); some had repeated measurements for a total of 1015 study visits. All participants had a blood draw to obtain baseline measures of lipopolysaccharide binding protein-a measure of intestinal permeability, as well as three inflammatory markers that were incorporated into an inflammatory index: C-reactive protein, interleukin-6, and tumor necrosis factor-α. They reported depressive symptoms on the Center for Epidemiological Studies depression scale (CES-D), and a binary variable indicated clinically significant depressive symptoms (CES-D ≥ 16). The Kohli (749 observations) and the Breast Cancer Prevention Trial (591 observations) scales assessed subjective cognitive function. Objective cognitive function tests included the trail-making test, Hopkins verbal learning test, Conners continuous performance test, n-back test, FAS test, and animal-naming test (239-246 observations). Adjusting for education, age, BMI, cancer treatment type, time since treatment, study visit, and fatigue, women who had clinically elevated depressive symptoms accompanied by heightened inflammation or intestinal permeability reported poorer focus and marginally poorer memory. However, poorer performance across objective cognitive measures was not specific to inflammation-associated depression. Rather, there was some evidence of lower verbal fluency; poorer attention, verbal learning and memory, and working memory; and difficulties with visuospatial search among depressed survivors, regardless of inflammation. By themselves, inflammation and intestinal permeability less consistently predicted subjective or objective cognitive function. Breast cancer survivors with clinically significant depressive symptoms accompanied by either elevated inflammation or intestinal permeability may perceive greater cognitive difficulty, even though depression-related objective cognitive deficits may not be specific to inflammation- or leaky-gut-associated depression.

ICAM-1-decorated extracellular vesicles loaded with miR-146a and Glut1 drive immunomodulation and hinder tumor progression in a murine model of breast cancer.

Tumor-associated immune cells play a crucial role in cancer progression. Myeloid-derived suppressor cells (MDSCs), for example, are immature innate immune cells that infiltrate the tumor to exert immunosuppressive activity and protect cancer cells from the host's immune system and/or cancer-specific immunotherapies. While tumor-associated immune cells have emerged as a promising therapeutic target, efforts to counter immunosuppression within the tumor niche have been hampered by the lack of approaches that selectively target the immune cell compartment of the tumor, to effectively eliminate "tumor-protecting" immune cells and/or drive an "anti-tumor" phenotype. Here we report on a novel nanotechnology-based approach to target tumor-associated immune cells and promote "anti-tumor" responses in a murine model of breast cancer. Engineered extracellular vesicles (EVs) decorated with ICAM-1 ligands and loaded with miR-146a and Glut1, were biosynthesized (in vitro or in vivo) and administered to tumor-bearing mice once a week for up to 5 weeks. The impact of this treatment modality on the immune cell compartment and tumor progression was evaluated via RT-qPCR, flow cytometry, and histology. Our results indicate that weekly administration of the engineered EVs (i.e., ICAM-1-decorated and loaded with miR-146a and Glut1) hampered tumor progression compared to ICAM-1-decorated EVs with no cargo. Flow cytometry analyses of the tumors indicated a shift in the phenotype of the immune cell population toward a more pro-inflammatory state, which appeared to have facilitated the infiltration of tumor-targeting T cells, and was associated with a reduction in tumor size and decreased metastatic burden. Altogether, our results indicate that ICAM-1-decorated EVs could be a powerful platform nanotechnology for the deployment of immune cell-targeting therapies to solid tumors.

BTK inhibition potentiates anti-PD-L1 treatment in murine melanoma: potential role for MDSC modulation in immunotherapy.

Myeloid-derived suppressor cells (MDSC) have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species and the production of inhibitory cytokines. Our group has shown that signaling through Bruton's tyrosine kinase (BTK) is important for MDSC function. Ibrutinib is an orally administered targeted agent that inhibits BTK activation and is currently used for the treatment of B cell malignancies. Using a syngeneic murine model of melanoma, the effect of BTK inhibition with ibrutinib on the therapeutic response to systemic PD-L1 blockade was studied. BTK was expressed by murine MDSC and their activation was inhibited by ibrutinib. Ibrutinib was not directly cytotoxic to cancer cells in vitro, but it inhibited BTK activation in MDSC and reduced expression of inducible nitric oxide synthase (NOS2) and production of nitric oxide. Ibrutinib treatments decreased the levels of circulating MDSC in vivo and increased the therapeutic efficacy of anti-PD-L1 antibody treatment. Gene expression profiling showed that ibrutinib decreased Cybb (NOX2) signaling, and increased IL-17 signaling (upregulating downstream targets Mmp9, Ptgs2, and S100a8). These results suggest that further exploration of MDSC inhibition could enhance the immunotherapy of advanced melanoma.PrécisInhibition of Bruton's tyrosine kinase, a key enzyme in myeloid cellular function, improves therapeutic response to an anti-PD-L1 antibody in an otherwise fairly resistant murine melanoma model.

IL-27 Gene Therapy Induces Stat3-Mediated Expansion of CD11b+Gr1+ Myeloid Cells and Promotes Accumulation of M1 Macrophages in the Tumor Microenvironment.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells and has a potential to be used as a therapeutic for cancer. We have recently demonstrated that administration of IL-27 producing adeno-associated virus (AAV-IL-27) exhibits potent inhibition of tumor growth in mouse models. In this study, we demonstrate that AAV-IL-27 treatment leads to significant expansion of CD11b+Gr1+ myeloid cells. AAV-IL-27-induced expansion of CD11b+Gr1+ cells is IL-27R-dependent and requires Stat3 signaling, but it is inhibited by Stat1 signaling. AAV-IL-27 treatment does not increase the self-renewal capacity of CD11b+Gr1+ cells but induces significant expansion of Lin-Sca1+c-Kit+ (LSK) and granulocyte-monocyte progenitor cells. Despite exhibiting significant suppression of T cells in vitro, IL-27-induced CD11b+Gr1+ cells lost the tumor-promoting activity in vivo and overall play an antitumor role. In tumors from AAV-IL-27-treated mice, CD11b+Gr1+ cells are largely F4/80+ and express high levels of MHC class I/II and M1 macrophage markers. Thus, IL-27 gene therapy induces Stat3-mediated expansion of CD11b+Gr1+ myeloid cells and promotes accumulation of M1 macrophages in the tumor microenvironment.

Expression of microRNAs and their target genes in melanomas originating from gynecologic sites.

Melanomas from gynecologic sites (MOGS) are rare and have poor survival. MicroRNAs (miRs) regulate gene expression and are dysregulated in cancer. We hypothesized that MOGS would display unique miR and mRNA expression profiles. The miR and mRNA expression profile in RNA from formalin fixed, paraffin embedded vaginal melanomas (relative to vaginal mucosa) and vulvar melanomas (relative to cutaneous melanoma) were measured with the Nanostring Human miRNA assay and Tumor Signaling mRNA assay. Differential patterns of expression were identified for 21 miRs in vaginal and 47 miRs in vulvar melanoma (fold change >2, p<0.01). In vaginal melanoma, miR-145-5p (tumor suppressor targeting TLR4, NRAS) was downregulated and miR-106a-5p, miR-17-5p, miR-20b-5p (members of miR-17-92 cluster) were upregulated. In vulvar melanoma, known tumor suppressors miR-200b-3p and miR-200a-3p were downregulated, and miR-20a-5p and miR-19b-3p, from the miR-17-92 cluster, were upregulated. Pathway analysis showed an enrichment of "proteoglycans in cancer". Among differentially expressed mRNAs, topoisomerase IIα (TOP2A) was upregulated in both MOGS. Gene targets of dysregulated miRs were identified using publicly available databases and Pearson correlations. In vaginal melanoma, suppressor of cytokine signaling 3 (SOCS3) was downregulated, was a validated target of miR-19b-3p and miR-20a-5p and trended toward a significant inverse Pearson correlation with miR-19b-3p (p = 0.093). In vulvar melanoma, cyclin dependent kinase inhibitor 1A (CDKN1A) was downregulated, was the validated target of 22 upregulated miRs, and had a significant inverse Pearson correlation with miR-503-5p, miR-130a-3p, and miR-20a-5p (0.005 < p < 0.026). These findings support microRNAs as mediators of gene expression in MOGS.

CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor-associated myeloid cells.

CD200 is overexpressed in many solid tumors and considered as an immune checkpoint molecule dampening cancer immunity. In this study, we found that CD200R-/- mice were significantly more potent in rejecting these CD200+ tumors. scRNA sequencing demonstrated that tumors from CD200R-/- mice had more infiltration of CD4+ and CD8+ T cells, and NK cells but less infiltration of neutrophils. Antibody depletion experiments revealed that immune effector cells are crucial in inhibiting tumor growth in CD200R-/- mice. Mechanistically, we found that CD200R signaling regulates the expression of chemokines in tumor-associated myeloid cells (TAMCs). In the absence of CD200R, TAMCs increased expression of CCL24 and resulted in increased infiltration of eosinophils, which contributes to anti-tumor activity. Overall, we conclude that CD200R signaling contributes to unfavorable TME through chemokine-dependent recruitment of immune suppressive neutrophils and exclusion of anti-cancer immune effectors. Our study has implications in developing CD200-CD200R targeted immunotherapy of solid tumors.

Association of Allostatic Load With All-Cause Mortality in Patients With Breast Cancer.

Importance: Elevated allostatic load (AL) has been associated with adverse socioenvironmental stressors and tumor characteristics that convey poor prognosis in patients with breast cancer. Currently, the association between AL and all-cause mortality in patients with breast cancer is unknown. Objective: To examine the association between AL and all-cause mortality in patients with breast cancer. Design, Setting, and Participants: This cohort study used data from an institutional electronic medical record and cancer registry at the National Cancer Institute Comprehensive Cancer Center. Participants were patients with breast cancer diagnoses (stage I-III) between January 1, 2012, through December 31, 2020. Data were analyzed from April 2022 through November 2022. Exposure: AL was expressed as a summary score calculated by assigning 1 point for biomarkers in the worst sample quartile. High AL was defined as AL greater than the median. Main Outcomes and Measures: The main outcome was all-cause mortality. A Cox proportional hazard models with robust variance tested the association between AL and all-cause mortality. Results: There were 4459 patients (median [IQR] age, 59 [49-67] years) with an ethnoracial distribution of 3 Hispanic Black patients (0.1%), 381 non-Hispanic Black patients (8.5%), 23 Hispanic White patients (0.5%), 3861 non-Hispanic White patients (86.6%), 27 Hispanic patients with other race (0.6%), and 164 non-Hispanic patients with other race (3.7%). The mean (SD) AL was 2.6 (1.7). Black patients (adjusted relative ratio [aRR], those with 1.11; 95% CI, 1.04-1.18), single marital status (aRR, 1.06; 95% CI, 1.00-1.12), and those with government-supplied insured (Medicaid aRR, 1.14; 95% CI, 1.07-1.21; Medicare aRR, 1.11; 95% CI, 1.03-1.19) had a higher adjusted mean AL than those who were White, married/living as married, or privately insured, respectively. Adjusting for sociodemographic, clinical, and treatment factors, high AL was associated with a 46% increase in mortality risk (hazard ratio [HR], 1.46; 95% CI, 1.11-1.93) over low AL. Similarly, compared with patients in the first AL quartile, those in the third quartile (HR, 1.53; 95% CI, 1.07-2.18) and the fourth quartile (HR, 1.79; 95% CI, 1.16-2.75) had significantly increased risks of mortality. There was a significant dose-dependent association between increased AL and a higher risk of all-cause mortality. Furthermore, AL remained significantly associated with higher all-cause mortality after adjusting for the Charlson Comorbidity Index. Conclusions and Relevance: These findings suggest increased AL is reflective of socioeconomic marginalization and associated with all-cause mortality in patients with breast cancer.