Identification of Hypothalamic Glucoregulatory Neurons That Sense and Respond to Changes in Glycemia.

To investigate whether glucoregulatory neurons in the hypothalamus can sense and respond to physiological variation in the blood glucose (BG) level, we combined continuous arterial glucose monitoring with continuous measures of the activity of a specific subset of neurons located in the hypothalamic ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) obtained using fiber photometry. Data were collected in conscious, free-living mice during a 1-h baseline monitoring period and a subsequent 2-h intervention period during which the BG level was raised either by consuming a chow or a high-sucrose meal or by intraperitoneal glucose injection. Cross-correlation analysis revealed that, following a 60- to 90-s delay, interventions that raise the BG level reliably associate with reduced VMNPACAP neuron activity (P < 0.01). In addition, a strong positive correlation between BG and spontaneous VMNPACAP neuron activity was observed under basal conditions but with a much longer (∼25 min) temporal offset, consistent with published evidence that VMNPACAP neuron activation raises the BG level. Together, these findings are suggestive of a closed-loop system whereby VMNPACAP neuron activation increases the BG level; detection of a rising BG level, in turn, feeds back to inhibit these neurons. To our knowledge, these findings constitute the first evidence of a role in glucose homeostasis for glucoregulatory neurocircuits that, like pancreatic ß-cells, sense and respond to physiological variation in glycemia. ARTICLE HIGHLIGHTS: By combining continuous arterial glucose monitoring with fiber photometry, studies investigated whether neurons in the murine ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) detect and respond to changes in glycemia in vivo. VMNPACAP neuron activity rapidly decreases (within <2 min) when the blood glucose level is raised by either food consumption or glucose administration. Spontaneous VMNPACAP neuron activity also correlates positively with glycemia, but with a longer temporal offset, consistent with reports that hyperglycemia is induced by experimental activation of these neurons. Like pancreatic ß-cells, neurons in the hypothalamic ventromedial nucleus appear to sense and respond to physiological variation in glycemia.

PAI-1 is a potential transcriptional silencer that supports bladder cancer cell activity.

The extracellular activity of Plasminogen activator inhibitor-1 (PAI-1) is well described, acting as an inhibitor of tissue plasminogen activator and urokinase-type plasminogen activator, impacting fibrinolysis. Recent studies have revealed a pro-tumorigenic role of PAI-1 in human cancers, via the regulation of angiogenesis and tumor cell survival. In this study, immunohistochemical staining of 939 human bladder cancer specimens showed that PAI-1 expression levels correlated with tumor grade, tumor stage and overall survival. The typical subcellular localization of PAI-1 is cytoplasmic, but in approximately a quarter of the cases, PAI-1 was observed to be localized to both the tumor cell cytoplasm and the nucleus. To investigate the potential function of nuclear PAI-1 in tumor biology we applied chromatin immunoprecipitation (ChIP)-sequencing, gene expression profiling, and rapid immunoprecipitation mass spectrometry to a pair of bladder cancer cell lines. ChIP-sequencing revealed that PAI-1 can bind DNA at distal intergenic regions, suggesting a role as a transcriptional coregulator. The downregulation of PAI-1 in bladder cancer cell lines caused the upregulation of numerous genes, and the integration of ChIP-sequence and RNA-sequence data identified 57 candidate genes subject to PAI-1 regulation. Taken together, the data suggest that nuclear PAI-1 can influence gene expression programs and support malignancy.

Phase Ib study of patients with metastatic castrate-resistant prostate cancer treated with different sequencing regimens of atezolizumab and sipuleucel-T.

BACKGROUND: Combining an immune checkpoint inhibitor with a tumor vaccine may modulate the immune system to leverage complementary mechanisms of action that lead to sustained T-cell activation and a potent prolonged immunotherapeutic response in metastatic castration resistant prostate cancer (mCRPC). METHODS: Subjects with asymptomatic or minimally symptomatic mCRPC were randomly assigned in a 1:1 ratio to receive either atezolizumab followed by sipuleucel-T (Arm 1) or sipuleucel-T followed by atezolizumab (Arm 2). The primary endpoint was safety, while secondary endpoints included preliminary clinical activity such as objective tumor response and systemic immune responses that could identify key molecular and immunological changes associated with sequential administration of atezolizumab and sipuleucel-T. RESULTS: A total of 37 subjects were enrolled. The median age was 75.0 years, median prostate specific antigen (PSA) was 21.9 ng/mL, and subjects had a median number of three prior treatments. Most subjects (83.8%) had at least one treatment-related adverse event. There were no grade 4 or 5 toxicities attributed to either study drug. Immune-related adverse events and infusion reactions occurred in 13.5% of subjects, and all of which were grade 1 or 2. Of 23 subjects with Response Evaluation Criteria in Solid Tumors measurable disease, only one subject in Arm 2 had a partial response (PR) and four subjects overall had stable disease (SD) at 6 months reflecting an objective response rate of 4.3% and a disease control rate of 21.7%. T-cell receptor diversity was higher in subjects with a response, including SD. Immune response to three novel putative antigens (SIK3, KDM1A/LSD1, and PIK3R6) appeared to increase with treatment. CONCLUSIONS: Overall, regardless of the order in which they were administered, the combination of atezolizumab with sipuleucel-T appears to be safe and well tolerated with a comparable safety profile to each agent administered as monotherapy. Correlative immune studies may suggest the combination to be beneficial; however, further studies are needed. TRIAL REGISTRATION NUMBER: NCT03024216.

Safety, Tolerability, and Long-Term Clinical Outcomes of an IL-15 analogue (N-803) Admixed with Bacillus Calmette-Guérin (BCG) for the Treatment of Bladder Cancer.

Intravesical BCG is active against non-muscle invasive bladder cancer (NMIBC), but bladder cancer will recur and even progress in a significant number of patients. To improve the response rate, N-803, an IL-15 superagonist was administered in combination with BCG. To evaluate the safety and efficacy associated with the use of intravesical N-803 and BCG in patients with BCG-naïve NMIBC. This phase 1b clinical trial used a 3 + 3 dose-escalation design. Participants were enrolled from July 2014 and July 2015, with follow-up and analyses through January 15, 2021. Eligibility criteria included histologically confirmed non-muscle invasive urothelial carcinoma of intermediate or high risk who had not received prior treatment with intravesical BCG (ie, BCG-naïve). All 9 participants met the eligibility criteria, received treatment according to the protocol, and were included in all analyses. Treatment was done once weekly for 6 consecutive weeks with bladder infusion of the standard dose of BCG, 50 mg/instillation, in combination with increasing doses of N-803 (100, 200, or 400 µg N-803 per instillation). No DLTs were noted in any of the dose cohorts. All adverse events (AEs) were manageable and less than grade 3. During the 2-year follow-up, all 9 participants were disease free. Furthermore, 6 y after treatment, all 9 participants (100%) were disease free with no evidence of disease progression and an intact bladder. This phase 1b trial found the combination of intravesical N-803 and BCG to be associated with modest toxic effects, low immunogenicity, and substantial prolonged antitumoral activity; phase 2 trials are in progress.

BRAFV600E, hypothyroidism, and human relaxin in thyroid carcinogenesis.

PURPOSE: BRAFV600E, a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin. METHODS: Immunohistochemical expressions of BRAFV600E, TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004. RESULTS: BRAFV600E was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAFV600E prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAFV600E mutation in classic papillary tumors significantly increased from 56 to 72% over the 21-year period of diagnoses, while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival, but did not remain significant after adjustment for demographic and clinical factors. CONCLUSIONS: Our study provides the first evidence of the potential interaction of BRAFV600E mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAFV600E in thyroid carcinogenesis. BRAFV600E mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy.

Breast tumor tissue inflammation but not lobular involution is associated with survival among breast cancer patients in the Multiethnic Cohort.

BACKGROUND: This study investigated the association of breast lobular involution status and three inflammatory markers as predictors of survival among breast cancer patients in the Multiethnic Cohort. METHODS: Lobular involution was evaluated in tissue sections of normal breast tissue and COX-2, TNF-α, and TGF-ß proteins were assessed by immunohistochemistry in tumor microarrays. A summary score added the expression levels of the three markers. Cox regression was applied to estimate hazard ratios (HRs) and 95 % confidence intervals (CI) with age as the time metric and adjustment for factors known to affect mortality. RESULTS: Among 254 women (mean age = 61.7 ± 8.7 years) with pathologic blocks and follow-up information, 54 all-cause and 10 breast cancer-specific deaths were identified after a mean follow-up time of 16.0 ± 3.1 years. For 214 participants, an inflammatory score was available and 157 women had information on lobular involution. Lobular involution was not significantly associated with survival. Expression of both COX-2 and TNF-α were significant predictors of lower survival (p = 0.02 and 0.04), while the association for TGF-ß was weaker (p = 0.09). When combined into one overall inflammation score, both intermediate (HR = 2.72; 95 % CI 0.90-8.28) and high (HR = 4.21; 95 % CI 1.51-11.8) scores were associated with higher mortality but only the latter was statistically significant. No significant association with breast cancer-specific mortality was detected. CONCLUSIONS: These results suggest that strong expression of inflammatory markers in breast tissue predicts a poorer prognosis possibly due to a system-wide state of chronic inflammation.

Plasminogen activator inhibitor-2 (PAI-2) overexpression supports bladder cancer development in PAI-1 knockout mice in N-butyl-N- (4-hydroxybutyl)-nitrosamine- induced bladder cancer mouse model.

BACKGROUND: Accumulating evidence suggests that plasminogen activator inhibitor-1 (PAI-1) plays an important role in bladder tumorigenesis by regulating cell cycle. However, it remains unclear whether and how inhibition of PAI-1 suppresses bladder tumorigenesis. METHODS: To elucidate the therapeutic effect of PAI-1 inhibition, we tested its tumorigenicity in PAI-1 knockout (KO) mice exposed to a known bladder carcinogen. RESULTS: PAI-1 deficiency did not inhibit carcinogen-induced bladder cancer in mice although carcinogen-exposed wild type mice significantly increased PAI-1 levels in bladder tissue, plasma and urine. We found that PAI-1 KO mice exposed to carcinogen tended to upregulate protein C inhibitor (PAI-3), urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA), and significantly increased PAI-2, suggesting a potential compensatory function of these molecules when PAI-1 is abrogated. Subsequent studies employing gene expression microarray using mouse bladder tissues followed by post hoc bioinformatics analysis and validation experiments by qPCR and IHC demonstrated that SERPING1 is further downregulated in PAI-1 KO mice exposed to BBN, suggesting that SERPING1 as a potential missing factor that regulate PAI-2 overexpression (compensation pathway). CONCLUSIONS: These results indicate that serpin compensation pathway, specifically PAI-2 overexpression in this model, supports bladder cancer development when oncoprotein PAI-1 is deleted. Further investigations into PAI-1 are necessary in order to identify true potential targets for bladder cancer therapy.

An Unlikely Route: Metastatic Ovarian Malignancy within the Duodenum.

The small bowel is an uncommon site for cancer metastasis. Despite this, cases have reported the duodenum as a metastatic site from local organs. However, duodenal involvement from more distant organs, such as the ovaries, has rarely been reported. Herein, we present a case of a 68-year-old female who developed duodenal metastatic disease from a primary ovarian serous adenocarcinoma. The goal of this report is to encourage clinicians to keep a broad differential in patients complaining of abdominal pain, especially in those with a history of primary ovarian malignancy.

Outlasting a Rare Duodenal Angiosarcoma.

With current life expectancies exceeding 78 years on average, to be confronted with the discovery of a rare cancer often found in advanced stages is a startling devastation. Angiosarcoma of the intestine is a rare and aggressive tumor that is not often considered in the differential diagnosis of intestinal obstruction. Once found and accurately diagnosed, it is a bewildering race against time as its median survival time is 150 days from diagnosis. This case report details a rare small intestinal angiosarcoma with its host surpassing current epidemiological standards of survival time despite only being eligible for chemotherapy.

Underlying liver disease and advanced stage liver cancer are associated with elevated neutrophil-lymphocyte ratio.

BACKGROUND/AIMS: Inflammation-based scores, such as the neutrophil-to-lymphocyte ratio (NLR), have been associated with prognosis in hepatocellular carcinoma (HCC); but variable cut-off values and potential lack of specificity have limited the utility of NLR. This study evaluates NLR in a large cohort of HCC patients. METHODS: We retrospectively reviewed 789 HCC cases (1993-2017) for demographics, tumor characteristics, treatment, and survival. NLR was stratified into NLR ≥1.5 and NLR ≥3 and analyzed for correlation with American Joint Committee on Cancer (AJCC) and Barcelona Clinic Liver Cancer (BCLC) stages. In 235 patients who underwent liver resection, survival and recurrence were evaluated by NLR. RESULTS: In 789 HCC cases, mean NLR was increased with advanced AJCC and BCLC stages. Hepatitis C patients were less likely to have NLR ≥1.5 and ≥3. Non-alcoholic steatohepatitis patients were more likely to have NLR ≥3. Patients with tumor size >5 cm, rupture, or macrovascular invasion were more likely to have NLR ≥3. In patients treated with resection, NLR ≥3 predicted early recurrence (odds ratio [OR] 4.14, P<0.01) and overall recurrence (OR 4.05, P<0.01). Mean NLR was 4.30 in those with recurrence and 2.75 in those without recurrence. Patients with NLR ≥3 showed significantly worse survival compared to those with NLR <3 (P<0.01 by log-rank test). CONCLUSION: Elevated NLR is associated with advanced cancer stage and aggressive tumor characteristics, such as large size, rupture, and invasion. NLR ≥3 was associated with early and overall recurrence after resection but varied with etiology. NLR may be a useful biomarker in predicting recurrence for HCC patients undergoing curative resection, but further studies are required to elucidate the effect of disease etiology.

Recurrent glucose deprivation leads to the preferential use of lactate by neurons in the ventromedial hypothalamus.

Increased GABAergic output in the ventromedial hypothalamus (VMH) contributes to counterregulatory failure in recurrently hypoglycemic (RH) rats, and lactate, an alternate fuel source in the brain, contributes to this phenomenon. The current study assessed whether recurring bouts of glucose deprivation enhanced neuronal lactate uptake and, if so, whether this influenced γ-aminobutyric acid (GABA) output and the counterregulatory responses. Glucose deprivation was induced using 5-thioglucose (5TG). Control rats received an infusion of artificial extracellular fluid. These groups were compared with RH animals. Subsequently, the rats underwent a hypoglycemic clamp with microdialysis. To test whether 5TG affected neuronal lactate utilization, a subgroup of 5TG-treated rats was microinjected with a lactate transporter inhibitor [cyano-4-hydroxycinnamate (4CIN)] just before the start of the clamp. Both RH and 5TG raised VMH GABA levels, and this was associated with impaired counterregulatory responses. 4CIN reduced VMH GABA levels and restored the hormone responses in the 5TG group. We then evaluated [14C]lactate uptake in hypothalamic neuronal cultures. Recurring exposure to low glucose increased monocarboxylate transporter-2 mRNA expression and augmented lactate uptake. Taken together, our data suggest that glucose deprivation, per se, enhances lactate utilization in hypothalamic neurons, and this may contribute to suppression of the counterregulatory responses to hypoglycemia.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Human papillomavirus DNA detection, p16INK4a, and oral cavity cancer in a U.S. population.

OBJECTIVES: The role of HPV in oral cavity cancers was investigated using two markers of viral exposure. MATERIALS AND METHODS: HPV DNA and p16INK4a expression were evaluated in tumor tissue from a U.S. population-based sample of 122 invasive oral cavity cancer cases. RESULTS: HPV DNA was detected in 38 of 122 (31%) oral cavity tumors. Seven genotypes were detected including HPV 16, which was found in 22% of tumors. p16INK4a was expressed in 30% of tumors and was poorly correlated with HPV DNA detection (Kappa <0.1). Joint positivity for HPV 16 and/or 18 and p16INK4a was observed in only 7% of cases. When comparing cases diagnosed in 1993-1999 and in 2000-2004, positivity for HPV DNA 16/18 increased from 19% to 39% (p = 0.02) and joint HPV 16/18 - p16INK4a positivity increased from 0% to 12% (p = 0.01). For gingival tumors, HPV 16 and/or 18 positivity was 67% compared to 11-38% for other sites (p = 0.02); joint HPV 16/18 - p16INK4a positivity was 33% compared to 0-8% for other sites (p = 0.01). The association of HPV with gingival tumors and more recent diagnosis period remained after adjustment for age and stage (p < 0.05). Neither HPV DNA nor p16INK4a were associated with overall survival. CONCLUSIONS: Based on both HPV DNA and p16INK4a, HPV is etiologically linked to a limited subset of oral cavity cancers. However, the role of HPV in oral cavity cancer may vary widely by subsite and may have increased over time, similar to trends observed for oropharyngeal cancer.

Monoclonal Antibody against CXCL1 (HL2401) as a Novel Agent in Suppressing IL6 Expression and Tumoral Growth.

Rationale: The expression of the chemokine (C-X-C motif) ligand 1 (CXCL1), an inflammatory protein, has been reported to be up-regulated in many human cancers. The mechanisms through which aberrant cellular CXCL1 levels promote specific steps in tumor growth and progression are unknown. Methods: We described the anticancer effects and mechanism of action of HL2401, a monoclonal antibody directed at CXCL1 with in vitro and in vivo data on bladder and prostate cancers. Results: HL2401 inhibited proliferation and invasion of bladder and prostate cells along with disrupting endothelial sprouting in vitro. Furthermore, novel mechanistic investigations revealed that CXCL1 expression stimulated interleukin 6 (IL6) expression and repressed tissue inhibitor of metalloproteinase 4 (TIMP4). Systemic administration of HL2401 in mice bearing bladder and prostate xenograft tumors retarded tumor growth through the inhibition of cellular proliferation and angiogenesis along with an induction of apoptosis. Our findings reveal a previously undocumented relationship between CXCL1, IL6 and TIMP4 in solid tumor biology. Principal conclusions: Taken together, our results argue that CXCL1 plays an important role in sustaining the growth of bladder and prostate tumors via up-regulation of IL6 and down-regulation of TIMP4. Targeting these critical interactions with a CXCL1 monoclonal antibody offers a novel strategy to therapeutically manage bladder and prostate cancers.

Effectiveness of two different dose administration regimens of an IL-15 superagonist complex (ALT-803) in an orthotopic bladder cancer mouse model.

BACKGROUND: We set out to determine if the administration of subcutaneous (SQ) ALT-803 was non-inferior to standard intravesical BCG treatment in a carcinogen induced mouse (C57BL/6J) bladder cancer model. METHODS: Using this well-established carcinogen induced mouse model, we studied the effects of various dosing schemas of ALT-803 (SQ alone, SQ with intravesical BCG, intravesical alone, intravesical with intravesical BCG) compared to intravesical BCG alone (positive control) and PBS (negative control). The non-inferiority margin for the difference in bladder weight, as a surrogate for tumor mass, was defined as 7%. RESULTS: All treatment groups (i.e., ALT-803 SQ alone, ALT-803 SQ with intravesical BCG, ALT-803 intravesical alone, ALT-803 intravesical with intravesical BCG and intravesical BCG alone) demonstrated a significant reduction in tumor burden as evident by bladder weights and H&E stain (p < 0.005). Non-inferiority tests between the intravesical BCG alone group and the additional treatment groups showed that SQ ALT-803 alone (p = 0.04) and BCG plus SQ ALT-803 (p = 0.009) were non-inferior to intravesical BCG alone. In this model, we did not see an appreciable infiltration of CD4+ T, CD8+ T or CD161/KLRB1+ natural killer (NK) cells in the bladder/tumor. When assessing peripheral blood mononuclear cells, SQ ALT-803 alone resulted in a robust induction of CD8+ T cells (p < 0.01), NKG2D+ NK cells (p < 0.005) and CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups, while in splenic tissue, SQ ALT-803 alone resulted in a robust induction of CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups. CONCLUSION: Subcutaneous ALT-803 treatment alone or in combination with intravesical BCG was well tolerated and was not inferior to intravesical BCG alone. CD8+ T, NKG2D+ NK and CD3+/NKG2D+ NKT cell induction along with induction of key cytokines remain steadfast mechanisms behind ALT-803. The enhanced therapeutic index seen with BCG and ALT-803, administered SQ or intravesically, provides a powerful justification for the further development of these regimens.

Prognostic Significance of Lymphocyte Infiltration and a Stromal Immunostaining of a Bladder Cancer Associated Diagnostic Panel in Urothelial Carcinoma.

We set out to expand on our previous work in which we reported the epithelial expression pattern of a urine-based bladder cancer-associated diagnostic panel (A1AT, ANG, APOE, CA9, IL8, MMP9, MMP10, PAI1, SDC1, and VEGFA). Since many of the analytes in the bladder cancer-associated diagnostic signature were chemokines, cytokines, or secreted proteins, we set out to report the stromal staining pattern of the diagnostic signature as well as CD3+ (T-cell) cell and CD68+ (macrophage) cell staining in human bladder tumors as a snapshot of the tumor immune landscape. Immunohistochemical staining was performed on 213 tumor specimens and 74 benign controls. Images were digitally captured and quantitated using Aperio (Vista, CA). The expression patterns were correlated with tumor grade, tumor stage, and outcome measures. We noted a positive correlation of seven of the 10 proteins (excluding A1AT and IL8 which had a negative association and VEGFA had no association) in bladder cancer. The overexpression of MMP10 was associated with higher grade disease, while overexpression of MMP10, PAI1, SDC1 and ANG were associated with high stage bladder cancer and CA9 was associated with low stage bladder cancer. Increased tumor infiltration of CD68+ cells were associated with higher stage disease. Overall survival was significantly reduced in bladder cancer patients' whose tumors expressed eight or more of the 10 proteins that comprise the bladder cancer diagnostic panel. These findings confirm that the chemokines, cytokines, and secreted proteins in a urine-based diagnostic panel are atypically expressed, not only in the epithelial component of bladder tumors, but also in the stromal component of bladder tumors and portends a worse overall survival. Thus, when assessing immunohistochemical staining, it is important to report staining patterns within the stroma as well as the entire stroma itself.

Pathology Characterization and Detection of Human Papillomavirus Type 16 in Rectal Squamous Cell Carcinomas.

Rectal squamous cell carcinoma (SCC) is a rare tumor with unresolved etiology. Human immunodeficiency virus-infected individuals and solid organ transplant recipients experience >30-fold and approximately 3-fold elevated rates of rectal SCC, respectively, suggesting immunosuppression plays a role.1 Human immunodeficiency virus-infected homosexual men have >60-fold higher rates of rectal SCC, similar to anal SCC. These patterns, which differ from the more common rectal adenocarcinoma (AdCA), raise the possibility of shared etiology between rectal and anal SCC, with human papillomavirus type 16 (HPV16) being a likely candidate.2.

Randomized controlled trial evaluating the utility of urine HPV DNA for cervical cancer screening in a Pacific Island population.

BACKGROUND: Non-invasive, self-collection sampling methods for human papillomavirus (HPV) DNA detection have the potential to address logistical and cultural barriers to Pap screening, particularly in under resourced settings such as Yap state in the Federated States of Micronesia - a population with low levels of screening and high incidence of cervical cancer. METHODS: A randomized controlled trial was conducted among adult women in Yap to compare cervical HPV DNA in self-collected urine and clinician-collected liquid cytology. Adult women aged 21-65 (n=217) were randomized by the order of sample collection. Concordance of HPV DNA, evaluated by the Roche Linear Array, was compared in paired self-collected urine and clinician-collected liquid cytology samples. The sensitivity and specificity of urine HPV DNA for prediction of cervical HPV and abnormal cytology was also evaluated. p16 in urine cytology samples was additionally assessed. RESULTS: Overall, HPV DNA detection was significantly lower in urine than cervical samples for any HPV (27.8% and 38.3%, respectively) and high-risk HPV (15.1% and 23.8%, respectively). For paired samples, there was moderate agreement for the overall study population (Kappa=0.54, 95% confidence interval CI=0.40-0.68) and substantial agreement for women >40 years (Kappa=0.65, 95% CI=0.46-0.85). The sensitivity and specificity of urine for the detection of cervical high-risk HPV was 51.0% and 96.2%, respectively. The sensitivities of HPV DNA in urine and liquid cytology for prediction of abnormal cytology (ASCUS/LSIL/HSIL) were 47.4% (95% CI=31.0-64.2) and 57.9% (95% CI=40.8-73.7), respectively; specificities were 92.0% (95% CI=86.9%-95.5%) and 83.5% (95% CI=77.2-88.7). Urine p16 was poorly correlated with urine HPV DNA positivity. CONCLUSIONS: Urine is less sensitive but more specific than directed cervical sampling for detection of cytologic abnormalities and may have utility for screening in older populations within low-resource communities when clinically-collected samples cannot be obtained.

Mammographic density and breast tissue expression of inflammatory markers, growth factors, and vimentin.

BACKGROUND: Mammographic density is a known risk factor for breast cancer, but the underlying pathologic characteristics are not well understood. The current analysis investigated the expression of several markers of interest, e.g., inflammation and growth, with mammographic density (MD) in normal and malignant breast tissue specimens from 279 women of the Multiethnic Cohort (MEC). METHODS: Breast cancer cases, recruited from a nested case-control study within the MEC, provided mammograms for density evaluation. Protein expression (COX-2, TNF-α, TGF-ß, IGF-1R, IGFBP-2, and vimentin) was assessed by immunohistochemical detection. Linear regression was applied to evaluate the relation between marker expression and percent density and to compute adjusted means with 95% confidence intervals (CI) by marker status while adjusting for confounders. RESULTS: Due to missing cores and tissue, normal tissue could only be evaluated for COX-2 and vimentin. No significant associations with mammographic density were detected for all markers analyzed. For inflammatory markers (TNF-α, COX-2, and TGF-ß) in tumor tissue, MD were non-significantly higher with stronger expression but the differences were very small. For example, the mean MD values for no, weak, and strong TNF-α expression were 35% (95% CI 24-47%), 39% (95% CI 29-48%), and 38% (95% CI 27-50%). In a posthoc analysis among postmenopausal women only, the difference across categories of TNF-α expression increased to 25% (95% CI 12-39%), 35% (95% CI 23-48%), and 35% (95% CI 20-49%). CONCLUSIONS: The current analysis offers little support for an involvement of immunohistochemical markers representing inflammatory and growth factor pathways as predictors of breast density.