Hyaluronan Staining Research Articles

Dysfunctional Extracellular Matrix Remodeling Supports Perianal Fistulizing Crohn′s Disease by a Mechanoregulated Activation of the Epithelial-to-Mesenchymal Transition

Perianal fistula represents one of the most disabling manifestations of Crohn's disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation. Surgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing. Genes significantly modulated were validated by real-time polymerase chain reaction, Western blot, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype, and ECM organization was investigated with endogenous lentivirus-induced overexpression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed. A markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and matrix metalloproteinases and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared with area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TNFAI6 expression and expression of mechanosensitive genes in fistula tissue. The overexpression of TSG-6 in Caco-2 cells promoted migration, epithelial-mesenchymal transition, transcription factor SNAI1, and HA synthase (HAs) levels, while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with recombinant human TSG-6 protein promoted collagen release and increase of SNAI1, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli. By mediating changes in the ECM organization, TSG-6 triggers the epithelial-mesenchymal transition transcription factor SNAI1 through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.

Stromal hyaluronan accumulation is associated with low immune response and poor prognosis in pancreatic cancer

Hyaluronan (HA) accumulation has been associated with poor survival in various cancers, but the mechanisms for this phenomenon are still unclear. The aim of this study was to investigate the prognostic significance of stromal HA accumulation and its association with host immune response in pancreatic ductal adenocarcinoma (PDAC). The study material consisted of 101 radically treated patients for PDAC from a single geographical area. HA staining was evaluated using a HA-specific probe, and the patterns of CD3, CD8, CD73 and PD-L1 expression were evaluated using immunohistochemistry. HA staining intensity of tumour stromal areas was assessed digitally using QuPath. CD3- and CD8-based immune cell score (ICS) was determined. High-level stromal HA expression was significantly associated with poor disease-specific survival (p = 0.037) and overall survival (p = 0.013) In multivariate analysis, high-level stromal HA expression was an independent negative prognostic factor together with histopathological grade, TNM stage, CD73 positivity in tumour cells and low ICS. Moreover, high-level stromal HA expression was associated with low ICS (p = 0.017). In conclusion, stromal HA accumulation is associated with poor survival and low immune response in PDAC.

Hyaluronan heterogeneity in pancreatic ductal adenocarcinoma, primary tumors, and sites of metastasis.

e16231 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with an estimated five-year survival rate of 10%. The dense desmoplastic stroma in PDAC contributes to its aggressive nature and treatment resistance. Among the components comprising the stroma, hyaluronan (HA) has been demonstrated to play a critical role in tumor progression and survival. Previous preliminary studies have suggested differences in HA expression in primary and metastatic foci in PDAC. However, the effects of treatment and location of HA expression as well as the role of CD44, a known receptor for HA, on HA as a biomarker signature remain unknown. Thus, we investigated the potential of HA as a biomarker in primary PDAC and metastases. Methods: PDAC tissue from primary (n = 43) and metastatic (n = 66) sites were obtained from Cedars-Sinai Medical Center along with associated clinical data. Tissue slides were stained with H&E, HA using a histochemical assay, and CD44 by immunohistochemistry. HA staining was scored according to the proportion of stromal staining at an intensity greater than the background stroma. HA status was defined as ≥ 50% staining being HA high and < 50% as being HA low. CD44 staining was recorded as an H-score (percentage of tumor cells staining multiplied by intensity of staining on a scale from 0 to 3). Associations between HA levels and the requested variables were examined with t-test, Wilcoxon rank-sum test, Chi-squared test, Fisher’s exact test, or Cox regression model where appropriate. Kaplan-Meier curves were created to assess progression free survival and overall survival. Analyses were performed using SAS 9.4 with two-sided tests and a significance level of 0.05. Results: HA score was significantly higher in primary PDAC tissue compared to sites of metastases (p = 0.0148). Within the metastases, HA score was significantly higher in liver metastasis compared to other sites of metastasis (p = 0.0478). In the liver metastasis tissue, HA score trended lower in patients with previously treated tissue compared to treatment naïve tissue (p = 0.0622). In the treatment naive liver metastasis cohort, patients with HA high status had decreased progression free survival and overall survival compared to patients with HA low status (p = 0.0032 and p = 0.0478, respectively). Using HA score and CD44 in a Cox regression model demonstrated that for every one unit increase in HA score, the risk for recurrence/progression increased by 4.4% at any fixed point in time, adjusting for CD44 score (p = 0.0049). Conclusions: HA score is variable between primary PDAC, PDAC metastatic to the liver, and PDAC metastatic to other sites. Within liver metastases, patients with HA high status had decreased progression free survival and overall survival compared to patients with HA low status. HA levels can serve as a potential biomarker to guide pancreatic cancer treatments and trial design for agents targeting the stroma.

The Vbeta13 T Cell Receptor Monoclonal Antibody Reduces Hyaluronan and CD68+, CD3+, and CD8+ Cell Infiltrations to Delay Diabetes in Congenic BB DRLyp/Lyp Rats.

The depleting Vβ13a T cell receptor monoclonal antibody (mAb) 17D5 prevents both induced and spontaneous autoimmune diabetes in BB rats. Here it was tested in congenic DRLyp/Lyp rats, all of which spontaneously developed diabetes. Starting at 40 days of age, rats were injected once weekly with either saline, His42 Vβ16 mAb, or 17D5 mAb and monitored for hyperglycemia. Diabetes occurred in 100% (n = 5/5) of saline-treated rats (median age, 66 days; range 55–73), and in 100% (n = 6/6) of His42-treated rats (median age, 69 days; range 59–69). Diabetes occurred in fewer (n = 8/11, 73%) 17D5-treated rats at a later age (median 76 days, range 60–92). Three (27%) of the 17D5-treated rats were killed at 101–103 days of age without diabetes (17D5 no-diabetes rats). Survival analysis demonstrated that 17D5 mAb delayed diabetes onset. Saline- and His42-treated rats had severely distorted islets with substantial loss of insulin-positive cells. These rats exhibited prominent hyaluronan (HA) staining, with the intra-islet HA+ accumulations measuring 5,000 ± 2,400 µm2 and occupying 36 ± 12% of islet area, and severe (grade 4) insulitis with abundant infiltration by CD68+, CD3+, and CD8+ cells. The 17D5 mAb-treated rats with delayed diabetes onset exhibited less severe insulitis (predominantly grade 3). In contrast, the 17D5 no-diabetes rats had mostly normal islets, with insulin+ cells representing 76 ± 3% of islet cells. In these rats, the islet HA deposits were significantly smaller than in the diabetic rats; the intra-islet HA+ areas were 1,200 ± 300 µm2 and accounted for 8 ± 1% of islet area. Also, islet-associated CD68+ and CD3+ cells occurred less frequently (on average in 60 and 3% of the islets, respectively) than in the diabetes rats (present in >95% of the islets). No CD8+ cells were detected in islets in all 17D5 no-diabetes rats. We conclude that mAb 17D5 delayed diabetes in DRLyp/Lyp rats and markedly reduced expression of HA and concomitant infiltration of CD68+, CD3+, and CD8+ cells. Our findings underscore the importance of refining immune suppression in prevention or intervention clinical trials to use mAb reagents that are directed against specific T cell receptors.

Vulnerability to shear stress caused by altered peri-endothelial matrix is a key feature of Moyamoya disease

Moyamoya disease (MMD) is characterized by progressive bilateral stenotic changes in the terminal portion of the internal carotid arteries. Although RNF213 was identified as a susceptibility gene for MMD, the exact pathogenesis remains unknown. Immunohistochemical analysis of autopsy specimens from a patient with MMD revealed marked accumulation of hyaluronan and chondroitin sulfate (CS) in the thickened intima of occlusive lesions of MMD. Hyaluronan synthase 2 was strongly expressed in endothelial progenitor cells in the thickened intima. Furthermore, MMD lesions showed minimal staining for CS and hyaluronan in the endothelium, in contrast to control endothelium showing positive staining for both. Glycosaminoglycans of endothelial cells derived from MMD and control induced pluripotent stem cells demonstrated a decreased amount of CS, especially sulfated CS, in MMD. A computational fluid dynamics model showed highest wall shear stress values in the terminal portion of the internal carotid artery, which is the predisposing region in MMD. Because the peri-endothelial extracellular matrix plays an important role in protection, cell adhesion and migration, an altered peri-endothelial matrix in MMD may contribute to endothelial vulnerability to wall shear stress. Invading endothelial progenitor cells repairing endothelial injury would produce excessive hyaluronan and CS in the intima, and cause vascular stenosis.

A Preclinical Investigation into the Effects of Aging on Dermal Hyaluronan Properties and Reconstitution Following Recombinant Human Hyaluronidase PH20 Administration.

IntroductionThere is currently no consensus in the literature concerning the impact of aging on the properties of hyaluronan (HA) in the subcutaneous (SC) space. Recombinant human hyaluronidase PH20 (rHuPH20) facilitates SC administration of injected therapeutics by depolymerizing SC HA, facilitating bulk fluid flow, dispersion and absorption. This study assessed the impact of intrinsic aging on HA in the SC space and thus the ability of rHuPH20 to enhance delivery of co-administered therapeutics.MethodsHistologic evaluations of HA levels and degradation were performed on human skin samples from six age groups, aged from 20 to 100 years. HA levels were evaluated by HA staining and degradation by staining samples for HA following incubation with rHuPH20. HA was extracted from samples and HA size determined by gel electrophoresis. Dermal reconstitution was assessed in young (aged 1.5 months) and elderly (aged > 16 months) mice. Baseline dye dispersion was measured at 5 and 20 min post-intradermal dye injection. Following treatment with rHuPH20, dye dispersion was measured again at 2, 24, 48, 72 and 96 h.ResultsDistribution of HA was confined to the interstitial space between adipocytes, with similar pericellular presence and levels of HA found across all age groups. Substantial levels of high-molecular-weight HA were observed in all age groups at baseline. Incubation with a clinically relevant dose of rHuPH20 resulted in degradation of all SC HA and similar degradation profiles independent of age. No difference in dye dispersion time was observed between young and elderly mice across the range of time points assessed, with dye dispersion returning to baseline levels by 24 h after rHuPH20 treatment.ConclusionsSubcutaneous delivery of approved therapeutics facilitated by co-administration with rHuPH20 should not be impacted by intrinsic aging, with this study providing no evidence for an effect of aging on HA distribution, structure or a loss of rHuPH20 efficacy.

Hyaluronan deposition in islets may precede and direct the location of islet immune-cell infiltrates

Substantial deposition of the extracellular matrix component hyaluronan (HA) is characteristic of insulitis in overt type 1 diabetes. We investigated whether HA accumulation is detectable in islets early in disease pathogenesis and how this affects the development of insulitis and beta cell mass. Pancreas tissue from 15 non-diabetic organ donors who were positive for islet autoantibodies (aAbs) and from 14 similarly aged aAb- control donors were examined for the amount of islet HA staining and the presence of insulitis. The kinetics of HA deposition in islets, along with the onset and progression of insulitis and changes in beta cell mass, were investigated in BioBreeding DRLyp/Lyp rats (a model of spontaneous autoimmune diabetes) from 40days of age until diabetes onset. Abundant islet HA deposits were observed in pancreas tissues from n = 3 single- and n = 4 double-aAb+ donors (aAb+HAhigh). In these seven tissues, the HA-stained areas in islets measured 1000 ± 240μm2 (mean ± SEM) and were fourfold larger than those from aAb- control tissues. The aAb+HAhigh tissues also had a greater prevalence of islets that were highly rich in HA (21% of the islets in these tissues contained the largest HA-stained areas [>2000μm2] vs less than 1% in tissues from aAb- control donors). The amount of HA staining in islets was associated with the number of aAbs (i.e. single- or double-aAb positivity) but not with HLA genotype or changes in beta cell mass. Among the seven aAb+HAhigh tissues, three from single- and one from double-aAb+ donors did not show any islet immune-cell infiltrates, indicating that HA accumulates in aAb+ donors independently of insulitis. The three aAb+HAhigh tissues that exhibited insulitis had the largest HA-stained areas and, in these tissues, islet-infiltrating immune cells co-localised with the most prominent HA deposits (i.e. with HA-stained areas >2000μm2). Accumulation of HA in islets was evident prior to insulitis in 7-8-week-old presymptomatic DRLyp/Lyp rats, in which the islet HA-stained area measured 2370 ± 170μm2 (mean ± SEM), which was threefold larger than in 6-week-old rats. This initial islet HA deposition was not concurrent with beta cell loss. Insulitis was first detected in 9-10-week-old rats, in which the HA-stained areas were 4980 ± 500μm2. At this age, the rats also exhibited a 44% reduction in beta cell mass. Further enlargement of the HA-positive areas (mean ± SEM: 7220 ± 880μm2) was associated with invasive insulitis. HA deposits remained abundant in the islets of rats with destructive insulitis, which had lost 85% of their beta cells. This study indicates that HA deposition in islets occurs early in type 1 diabetes and prior to insulitis, and points to a potential role of HA in triggering islet immune-cell infiltration and the promotion of insulitis.

506P - Efficacy and safety of pegvorhyaluronidase alfa (PEGPH20; PVHA) and pembrolizumab (pembro) combination therapy in patients (Pts) with stage III/IV non-small cell lung cancer (NSCLC)

BackgroundImmunotherapy is a promising treatment option for advanced NSCLC; however, its efficacy may be limited by accumulation of hyaluronan (HA). PEGPH20 degrades tumor-associated HA and increases the efficacy of chemo- and immuno-therapeutic agents in animal models. Here we report efficacy and safety results of PEGPH20 plus pembro in a cohort of pts with Stage IIIB/IV NSCLC in the 2-cohort, phase Ib dose escalation (DE) and expansion (DX) HALO 107-101 study (NCT02563548). MethodsThis analysis included pts with advanced NSCLC and ≥1 prior platinum-based chemotherapy (DE: 1.6 µg/kg and 2.2 µg/kg of PEGPH20, 2 pts per dose) and pts with HA-high NSCLC (HA staining ≥25% of tumor surface), previously untreated or treated with 1 prior platinum-based chemotherapy (DX: 17 pts). PEGPH20 was given on days 1, 8, and 15 and pembro (DE = 2 mg/kg; DX = 200 mg) on day 1 of 21day cycles. Pts received enteric-coated aspirin (81 mg daily) for thromboembolism prophylaxis. Primary endpoints included: DE: doselimiting toxicity, recommended phase 2 dose (RP2D); DX: objective response rate (ORR). Secondary endpoints included: ORR (DE only), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. ResultsMean pt age was 68 years; 76% of pts had an ECOG PS 1, and 52% were women. The RP2D dose of PEGPH20 was 2.2 μg/kg. A total of 17 of 21 enrolled pts had evaluable tumor responses: 4 had a partial response, 8 had stable disease, and 5 had progressive disease. ORR was 24% and DCR 71%. PFS rates at 6, 12, and 18 months were 25%, 0%, and 0%, respectively, and OS rates were 62%, 47%, and 31%, respectively. Any-grade treatment-emergent adverse events (TEAEs) were reported in all pts, the most common events being muscle spasms (71%), myalgia (43%), nausea (38%), fatigue (29%), and peripheral edema (29%). Grade ≥3 TEAEs were reported in 57% of pts; 38% of pts experienced ≥1 serious adverse event (AE). There was 1 (5%) treatment-emergent thromboembolic AE. ConclusionsThe combination of PEGPH20 with pembro has an acceptable safety profile that is consistent with results from other PEGPH20 clinical trials and demonstrates activity in pts with locally advanced or metastatic NSCLC. Clinical trial identificationNCT02563548. Editorial acknowledgementMedical writing support provided by Miriam Cohen, PhD, of Paragon Medica, Knutsford, UK, supported by Halozyme Therapeutics Inc. Legal entity responsible for the studyHalozyme Therapeutics Inc. FundingHalozyme Therapeutics Inc. DisclosureE. Bertino: Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy: Takeda. V. Lin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Halozyme Therapeutics Inc. V. Kuruvadi: Full / Part-time employment: Halozyme Therapeutics Inc. T. Heineman: Leadership role, Shareholder / Stockholder / Stock options: Halozyme Therapeutics Inc. All other authors have declared no conflicts of interest.

CD44s Assembles Hyaluronan Coat on Filopodia and Extracellular Vesicles and Induces Tumorigenicity of MKN74 Gastric Carcinoma Cells.

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far. To answer this question, we utilized CD44-negative human gastric carcinoma cell line MKN74 manipulated to stably express CD44 standard form (CD44s). The effect of CD44s expression on HA metabolism, EV secretion, morphology and growth of these cells was studied. Interestingly, HAS2 and HYAL2 expression levels were significantly upregulated in CD44s-expressing cells. Cell-associated HA levels were significantly increased, while HA levels in the culture medium of CD44s-positive cells was lower compared to CD44s-negative MOCK cells. CD44s expression had no significant effect on the proliferation capacity of cells, but cells showed diminished contact inhibition. Superresolution imaging revealed that CD44s and HA were accumulated on filopodia and EVs secreted from CD44s-positive cells, but no differences in total numbers of secreted EV between CD44s-negative and -positive cells was detected. In 3D cultures, CD44s-expressing cells had an enhanced invasion capacity in BME gel and increased spheroidal growth when cultured in collagen I gel. No significant differences in mitotic activity, tumor size or morphology were detected in CAM assays. However, a significant increase in HA staining coverage was detected in CD44s-positive tumors. Interestingly, CD44s-positive EVs embedded in HA-rich matrix were detected in the stromal areas of tumors. The results indicate that CD44s expression significantly increases the HA binding capacity of gastric cancer cells, while the secreted HA is downregulated. CD44s is also carried by EVs secreted by CD44s-expressing cells. These findings highlight the potential usefulness of CD44s and its ligands as multipurpose EV biomarkers, because they are upregulated in inflammatory, injured, and cancer cells and accumulate on the surface of EVs secreted in these situations.

Hyaluronidase inhibits reactive adipogenesis and inflammation of colon and skin

In this study we evaluated the role of hyaluronan (HA) in reactive adipogenesis, a local expansion of preadipocytes that provides host defense by release of antimicrobial peptides. We observed that HA accumulated during maturation of adipocytes in vitro and was associated with increased expression of preadipocyte factor 1, zinc finger protein 423, and early B cell factor 1. Although HA is normally abundant in the extracellular matrix, a further increase in HA staining occurred in mice at sites of reactive adipogenesis following injury of colon by dextran sodium sulfate or injury of skin from infection with Staphylococcus aureus. HA also abundantly accumulated around adipocytes seen in the colons of patients with inflammatory bowel disease. This HA was necessary for adipocyte maturation because digestion of HA by administration of soluble hyaluronidase or transgenic expression of hyaluronidase 1 inhibited adipogenesis in vitro and in vivo. Furthermore, hyaluronidase also suppressed inflammation of both skin and colon and decreased antimicrobial peptide expression by developing preadipocytes. This resulted in increased bacterial transit across the epithelial barrier despite decreased tissue injury from inflammation. These observations suggest HA plays an important role in reactive adipogenesis and host defense after injury.

Hyaluronan in vocal folds and false vocal folds in patients with recurrent respiratory papillomatosis

Background: Hyaluronan (HA) is a glycosaminoglycan with viscoelastic properties necessary for vocal fold (VF) vibration and voice production. Changes in HAs molecular mass, possibly related to human papilloma virus, could affect formation/persistence of recurrent respiratory papillomatosis (RRP). Aims/Objective: Describing mass and localization of HA and localization of HA receptor CD44 in VF and false vocal folds (FVF) in RRP. Materials and Methods: Biopsies from VF and FVF from 24 RRP patients. Twelve were studied with histo-/immunohistochemistry for HA and CD44 in epithelium, stroma and RRP lesions. Twelve samples were analyzed for HA molecular mass distribution with gas-phase-electrophoretic-molecular-mobility-analyzer (GEMMA). Results: Three of 23 stains (VF and FVF combined) showed faint HA staining in the epithelium; there was more extensive staining in the stroma. CD44 was present throughout all areas in FVF and VF, it did not concur with HA. GEMMA analysis revealed very high mass HA (vHMHA) with more varying amounts in VF. Conclusions/Significance: HA was mainly distributed in the stroma. CD44 not binding to HA might explain the non-inflammatory response described in RRP. Possibly crosslinked vHMHA was seen in VF and FVF, with more variable amounts in VF samples. Counteracting HA crosslinking could become a treatment option in RRP.

Transcriptomic Analysis Reveals Novel Mechanisms Mediating Islet Dysfunction in the Intrauterine Growth-Restricted Rat

Intrauterine growth restriction (IUGR) increases the risk of type 2 diabetes developing in adulthood. In previous studies that used bilateral uterine artery ligation in a rat model of IUGR, age-associated decline in glucose homeostasis and islet function was revealed. To elucidate mechanisms contributing to IUGR pathogenesis, the islet transcriptome was sequenced from 2-week-old rats, when in vivo glucose tolerance is mildly impaired, and at 10 weeks of age, when rats are hyperglycemic and have reduced β-cell mass. RNA sequencing and functional annotation with Ingenuity Pathway Analysis revealed temporal changes in IUGR islets. For instance, gene expression involving amino acid metabolism was significantly reduced primarily at 2 weeks of age, but ion channel expression, specifically that involved in cell-volume regulation, was more disrupted in adult IUGR islets. Additionally, we observed alterations in the microenvironment of IUGR islets with extracellular matrix genes being significantly increased at 2 weeks of age and significantly decreased at 10 weeks. Specifically, hyaluronan synthase 2 expression and hyaluronan staining were increased in IUGR islets at 2 weeks of age (P < 0.05). Mesenchymal stromal cell-derived factors that have been shown to preserve islet allograft function, such as Anxa1, Cxcl12, and others, also were increased at 2 weeks and decreased in adult islets. Finally, comparisons of differentially expressed genes with those of type 2 diabetic human islets support a role for these pathways in human patients with diabetes. Together, these data point to new mechanisms in the pathogenesis of IUGR-mediated islet dysfunction in type 2 diabetes.

Reduction of hyaluronan and increased expression of HYBID (alias CEMIP and KIAA1199) correlate with clinical symptoms in photoaged skin

Hyaluronan (HA) metabolism in skin fibroblasts is mediated by HYBID (hyaluronan binding protein involved in hyaluronan depolymerization, alias CEMIP and KIAA1199) and the HA synthases HAS1 and HAS2. However, photoageing-dependent changes in HA and their molecular mechanisms, and the relationship between HA metabolism and clinical symptoms in photoaged skin remain elusive. We examined the amount, size and tissue distribution of HA and expression levels of HYBID, HAS1 and HAS2 in photoaged skin, and analysed their relationship with the degree of photoageing. Photoageing-dependent changes of HA were investigated by studying skin biopsies isolated from photoprotected and photoexposed areas of the same donors, and the relationships between HA and photoageing symptoms such as skin wrinkling and sagging were examined. Skin biopsy specimens showed that the amount and size of HA are decreased in photoexposed skin compared with photoprotected skin, and this was accompanied by increased expression of HYBID and decreased expression of HAS1 and HAS2. Histologically, HA staining in the papillary dermis was decreased in photoexposed skin, showing reverse correlation with HYBID expression. HYBID expression in the photoexposed skin directly correlated with skin roughness and sagging parameters, and the reduced HA staining in the papillary dermis in the photoexposed skin positively correlated with these symptoms. These data demonstrate that imbalance between HYBID-mediated HA degradation and HAS-mediated HA synthesis may contribute to enhanced HA catabolism in photoaged skin, and suggest that HYBID-mediated HA reduction in the papillary dermis is related to skin wrinkling and sagging of photoaged skin.

Relationship of hyaluronan and HYBID (KIAA1199) expression with roughness parameters of photoaged skin in Caucasian women.

Hyaluronan (HA) is an important constituent of extracellular matrix (ECM) in the skin, and HA degradation mediated by HYBID (KIAA1199) is suggested to be implicated in facial skin wrinkling in Japanese women. Ethnic difference in skin wrinkle formation is known between Caucasian and Japanese women, but no information is available for the relations of HA and HYBID expression levels with skin wrinkling in Caucasian women. The skin surface roughness at the eye corner of the Caucasian female subjects was measured, and the skin specimens biopsied from the same areas were subjected to microarray gene analysis, HA staining, and immunohistochemistry for HYBID. Among the ECM genes and those related to ECM metabolism, only HYBID expression levels positively correlated with the skin roughness parameters. When the skin sample groups with high expression of HYBID or low expression of HYBID were compared, the HA staining intensity and the ratio of HYBID-immunoreactive cells to total cells in the superficial dermis were significantly reduced and increased in the high-HYBID-expression group compared with the low-HYBID-expression group, respectively. Our data suggest that like Japanese women, HYBID-mediated reduction of HA in the superficial dermis is involved in the formation of wrinkles in Caucasian women.

Transcriptomic Analysis Reveals Novel Mechanisms Mediating Islet Dysfunction in the Intrauterine Growth-Restricted Rat.

Intrauterine growth restriction (IUGR) increases the risk of type 2 diabetes developing in adulthood. In previous studies that used bilateral uterine artery ligation in a rat model of IUGR, age-associated decline in glucose homeostasis and islet function was revealed. To elucidate mechanisms contributing to IUGR pathogenesis, the islet transcriptome was sequenced from 2-week-old rats, when in vivo glucose tolerance is mildly impaired, and at 10 weeks of age, when rats are hyperglycemic and have reduced β-cell mass. RNA sequencing and functional annotation with Ingenuity Pathway Analysis revealed temporal changes in IUGR islets. For instance, gene expression involving amino acid metabolism was significantly reduced primarily at 2 weeks of age, but ion channel expression, specifically that involved in cell-volume regulation, was more disrupted in adult IUGR islets. Additionally, we observed alterations in the microenvironment of IUGR islets with extracellular matrix genes being significantly increased at 2 weeks of age and significantly decreased at 10 weeks. Specifically, hyaluronan synthase 2 expression and hyaluronan staining were increased in IUGR islets at 2 weeks of age (P < 0.05). Mesenchymal stromal cell-derived factors that have been shown to preserve islet allograft function, such as Anxa1, Cxcl12, and others, also were increased at 2 weeks and decreased in adult islets. Finally, comparisons of differentially expressed genes with those of type 2 diabetic human islets support a role for these pathways in human patients with diabetes. Together, these data point to new mechanisms in the pathogenesis of IUGR-mediated islet dysfunction in type 2 diabetes.

N-cadherin and hyaluronan expression in head and neck squamous cell carcinoma, relation to patient outcomes

Background: Epithelial-mesenchymal transition (EMT) is regarded as an essential step for tumor invasion and metastasis. In squamous cell carcinoma of head and neck (HNSCC), N-Cadherin expression and its involvement in tumor progression remains a controversial topic.Aim of the study: The present study aimed to assess the expression of N-cadherin and HA in HNSCC and further study their relation to patients survival and outcomes.Material and methods: Fifty-eight retrospective selected cases of head and neck squamous carcinomas (HNSCCs) with available paraffin blocks. Complete clinico-pathological and follow-up data were recorded. Immune staining for N-cadherin and hyaluronan were done, also, we study the correlation of the results with patients survival data.Results: Squamous cell carcinoma islands demonstrated high N-cadherin expression in 55.2% and low expression in 44.8%. N-cadherin high expression was significantly (p &lt; .05) associated with large tumor sizes, advanced TNM clinical stage, increased incidence of recurrence and patient’s death. A significant correlation was recorded between the presence of neural invasion and N-cadherin expression (p = .004). Strong intensity of stromal HA was significantly (p &lt; .05) associated with an oral site, nodal metastasis, and higher TNM stage. Patients with high N-cadherin expression, diffuse hyaluronan, and strong stromal hyaluronanreaction had significantly lower DFS rates (p &lt; .05). High N-cadherin expression, diffuse hyaluronan immunoreactivity, and strong stromal hyaluronan reaction intensity had significantly lower OS rates (p &lt; .05).Conclusion: N-cadherin and hyaluronan could be important and promising biomarkers during surveillance of patients with HNSCC.

260TiP - Phase 3, randomized, double-blind, placebo-controlled study of PEGylated recombinant human hyaluronidase PH20 (PEGPH20)+nab-paclitaxel/gemcitabine in patients with previously untreated, hyaluronan-high, stage IV pancreatic ductal adenocarcinoma (PDA)

Background: Poor outcome in PDA is associated with stromal hyaluronan (HA) accumulation, which may compromise treatment access. In animal models, PEGPH20 degrades tumor HA. Key data from a Phase 2 study showed that PEGPH20 plus chemotherapy improved efficacy over chemotherapy alone in tumors retrospectively identified to accumulate HA (“HA-High”). The objectives of this phase 3 study are to compare efficacy and safety of standard-dose nab-paclitaxel (NAB) and gemcitabine (GEM) combined with PEGPH20 or placebo in patients with HA-High, previously untreated, stage IV PDA. Primary endpoints are progression-free survival (PFS) and overall survival (OS). Secondary endpoints are objective response rate, duration of response, and safety. Trial design: 420 patients ≥18 years with untreated HA-High metastatic PDA, ECOG PS 0-1 are randomized (stratified by North America/Europe/other) 2:1 to NAB 125 mg/m2 + GEM 1000 mg/m2 + PEGPH20 3.0 μg/kg or to NAB + GEM + placebo, respectively. Patients with HA-High tumors are prospectively identified by the co-developed VENTANA HA RxDx Assay, which identifies HA in the extracellular matrix. HA-High status (indicating patients who may achieve clinical benefit) was determined by Halozyme to be ≥ 50% HA staining based on clinical outcome data from a Phase 2 study. Treatment is provided in 4-week cycles (Wk 1-3, Wk 4 rest) until disease progression, unacceptable toxicity, death, or consent withdrawal. PEGPH20 or placebo is given twice weekly (Cycle 1) then weekly (Cycles 2+), NAB + GEM once weekly for all cycles. Dexamethasone is given before and after PEGPH20 to reduce treatment-related musculoskeletal symptoms and enoxaparin is given to minimize thromboembolic events. Tumor response will be assessed by an independent central imaging vendor using RECIST v1.1. Adverse events will be graded per NCI CTCAE v4.03. An independent Data Monitoring Committee will evaluate safety and efficacy (PFS and OS) data. Trial initiated Q12016. Clinical trial indentification: EudraCT 2015-004068-13; NCT02715804. Legal entity responsible for the study: Halozyme Therapeutics Funding: Halozyme Therapeutics Disclosure: D-Y. Oh: Research funding from AstraZeneca, Array BioPharma. Y-J. Bang: Research funding: AstraZeneca, Novartis, Roche, MSD, Merck Serano, Bayer, GSK, BMS, Pfizer, Eli Lilly, Boeringer-Ingelheim, MacroGenics, Boston Biomedical, FivePrime, CKD, Ono, Otsuka, Taiho, Takeda, BeiGene, Hanmi, Green Cross, Curis Ad board member: AstraZeneca, Novartis, Roche, Genentech, MSD, Pfizer, Bayer, BMS, Eli Lilly, Merck Serano, FivePrime, Merrimack, Taiho, Ono, ADC Therapeutics, GreenCross, Samyang Biopharm. E. Van Cutsem: Research funding: Halozyme. A. Hendifar: Consulting or Advisory Role: Xbiotech, Novartis (self) Research Funding: Ipsen, Halozyme (institution). L. Zheng: Employment: Roche, Stock or Other Ownership: Roche, Honoraria: Roche, Travel, Accommodations, Expenses: Roche. M. Ducreux: Honoraria/Consultation Fees: Roche, Celgene, Merck Serono, Amgen, Novartis, Sanofi, Pfizer, Lilly, Servier, Halozyme; Grants/Research funding: Roche, Chugai, Pfizer Spouse: Head of BU, Sandoz. W. Harris: Consulting or Advisory Role: Neotherma Oncology, Bayer (self); Research Funding: Halozyme, BMS, Exelixis, Arqule, Polaris, Medimmune, BTG (institution). P. Corrie: Honoraria: Roche, Novartis, Bristol Myers Squibb, Merck Sharpe & Dohme, Pierre Fabre Research funding: Celgene; Ad hoc honoraria for lectures/meeting presentations: Novartis, Celgene, Merck Sharpe & Dohme. T. Seery: Consulting or Advisory Role: Bayer (self) Speakers' Bureau: Ipsen, Bayer (self). D. Chondros: Employment: Halozyme (self) Leadership: Halozyme (self) Stock or Other Ownership: Halozyme, Roche (self) Travel Accommodations, Expenses: Halozyme, Roche/Genentech (self). A. Bullock: Consulting or advisory board participation support: Halozyme, Celgene, and EMD Serono (self). All other authors have declared no conflicts of interest.

743P - Tumor hyaluronan (HA) is a novel biomarker: Results of the randomized phase 2 HALO 202 study of PEGPH20 plus nab-paclitaxel/gemcitabine (PAG) vs AG in previously untreated, metastatic pancreatic ductal adenocarcinoma (mPDA)

Background: PEGPH20 (P) degrades HA in the tumor microenvironment to increase access and therapeutic index of anticancer agents. In Stage 1 of this study, Halozyme Therapeutics, Inc. and Ventana Medical Systems, Inc., co-developed a novel HA assay, scoring algorithm, and cut-point, and showed improved progression-free survival (PFS) and objective response rate (ORR) in HA-High patients (pts) with PAG vs AG. Due to an imbalance in thromboembolic (TE) events in the PAG arm, the protocol was amended to add enoxaparin and exclude pts at high risk for TE events in Stage 2, which prospectively validated the algorithm and cut-point for the VENTANA HA RxDx assay. Methods: In Stage 2, pts with mPDA were randomized 2:1 to PAG (P 3 µg/kg IV 2x/wk x 3 wk [C1], then 1x/wk x 3 wk [C2+] + AG) vs AG every 28 days. Endpoints were: primary—PFS and TE events; secondary—PFS by HA level, ORR; OS by HA level was exploratory. Tumor HA was evaluated using the VENTANA HA RxDx assay and algorithm; HA-High was defined by HA staining in the extracellular matrix ≥50% of the entire tumor surface at any intensity. Results: 133 pts were enrolled; 125 pts were treated. As of December 16, 2016, an improvement in median PFS (91%) and median OS (50%) was observed in HA-High pts treated with PAG vs AG (Table), supporting tumor HA as a predictive marker for PEGPH20 efficacy. Among AG-treated pts, those with HA-High tumors showed poorer median PFS and median OS outcomes, suggesting that targeting tumor HA with PEGPH20 may improve the standard of care in mPDA.Table743PPAGAGHR (95% CI)HA-Highn = 24n = 11PFS, months8.64.50.63 (0.21-1.93)OS, months11.77.80.52 (0.22-1.23)HA-Lown = 53n = 23PFS, months6.07.21.21 (0.63-2.30)OS, months11.910.20.69 (0.38-1.23) Open table in a new tab Conclusions: This is the first randomized Phase 2 study evaluating and supporting tumor HA as a potential predictive biomarker informing patient selection for PEGPH20 treatment, based on improvement in both PFS and OS in HA-High pts. The results provide support for the ongoing phase 3 HALO 109-301 study with co-primary endpoints of PFS and OS. NCT01839487. Clinical trial identification: NCT01839487 Legal entity responsible for the study: Halozyme Therapeutics, Inc. Funding: Halozyme Therapeutics, Inc. Disclosure: A. Hendifar: Consulting or advisory role: Genentech, Novartis, Ipsen, Perthera. Travel, accommodations, expenses: Halozyme. A. Bullock: Consulting or advisory board participation support: Halozyme, Celgene, EMD Serono. T. Seery: Consulting or advisory role: Bayer. Speakers' Bureau: Ipsen, Bayer. L. Zheng: Consultancy or advisory role: Merrimack. Patents, royalties, other intellectual property: GVAX. Licensed to Aduro Biotech. Stock and other ownership: Z&L Medical Intl. Research funding: Bristol-Myers Squibb, Amgen, Iteos Therap, Gradalis, Merck, Halozyme. D. Sigal: Stock or other ownership: Novartis, BMS, Ignyta, Halozyme. Honoraria: Novartis, Serond, Halozyme. Speakers' Bureau: Novartis, Celgene, Bayer. Research funding: Halozyme. F.S. Braiteh: Author has disclosures to report for: Honoraria, Consulting or Advisory Role,Speaker's Bureau,Travels, Accommodation, Expenses. M. Zalupski: Research funding: Halozyme, OncoMed, Newlink Genetics. N. Bahary: Consulting or advisory role: Bayer/Onyx, EMD Serono. Research funding: New Links Genetics. W. Harris: Consulting or advisory role: Neotherma Oncology, Bayer. Research funding: Halozyme, BMS, Exelixis, Argule, Polaris, Medimmune, BTG. J. Pu: Employment: Roche; Research funding: Roche. Patents, royalties, other intellectual property: Roche (for author and institution). F. Lian: Stock or other ownership: Merck (Immediate Family Member). J. Zhu: Employment: Roche. Stock or other ownership: Roche. Honoraria: Roche. Travel, accommodations, expenses: Roche. W. Wu: Employment: Halozyme. Stock or other ownership: Halozyme. D. Chondros: Employee of Halozyme Therapeutics. P. Jiang: Employment: Halozyme. Stock or other ownership: Halozyme Therapeutics Inc. Patents, royalties, other intellectual property: Halozyme Therapeutics Inc. S.R. Hingorani: Consulting or advisory role: Halozyme, Aduro Biotech. Research funding to institution: Halozyme. All other authors have declared no conflicts of interest.

Abstract CT032: A Phase 1b study of PEGPH20 plus pembrolizumab in patients with selected hyaluronan-high solid tumors

Abstract Background: Hyaluronan (HA) accumulation in the tumor microenvironment increases tumor interstitial fluid pressure, promoting vascular collapse and limiting access of chemotherapy and immune cells to tumor sites. In animal models, HA-High tumors exhibit increased growth and metastasis, treatment resistance, and reduced survival. PEGPH20 is a PEGylated recombinant human hyaluronidase that enzymatically degrades tumor HA. Pembrolizumab (PEM) is a humanized monoclonal antibody targeting PD-1, demonstrating tolerability and activity in patients (pts) with non-small cell lung cancer (NSCLC). This study evaluates the safety and activity of PEGPH20 plus PEM in pts with NSCLC or gastric cancer. Methods: This Phase 1b study comprises dose escalation (up to 30 pts regardless of HA status) and cohort expansion (up to 51 pts with tumors accumulating high levels of HA). HA staining is performed using the VENTANA HA RxDX Assay, which indicates HA in the extracellular matrix. HA-High status (ie, patients who may achieve clinical benefit) was determined by Halozyme to be ≥50% staining based on clinical outcome data from a phase 2 study. For dose escalation, eligible pts (ECOG 0-1) had stage IIIB/IV NSCLC failing ≥1 prior platinum-based regimen or locally advanced or metastatic gastric adenocarcinoma failing ≥1 previous chemotherapy regimen. NSCLC pts known to be epidermal growth factor receptor (EGFR)- or anaplastic lymphoma kinase (ALK)-positive must have received an EGFR inhibitor or ALK inhibitor, respectively. For cohort expansion, previously treated or untreated NSCLC is allowed; gastric cancer pts must have failed 1-2 prior treatments. PEGPH20 (1.6, 2.2, 2.6, 3.0, 4.0 µg/kg) is administered IV on D1, 8, 15 of each 21-day cycle followed by PEM 200 mg IV on D1, 4 to 6 hours after PEGPH20. Due to increased thromboembolic risk in gastric cancer, these pts receive prophylactic enoxaparin. Additional prophylaxis with piroxicam (possible musculoskeletal events) and proton pump inhibitors are given to all patients. The primary endpoint for dose escalation is the recommended Phase 2 dose for PEGPH20 in combination with PEM. The primary endpoint for cohort expansion is objective response rate per RECIST v1.1. Secondary endpoints are duration of response, disease control rate, progression-free survival per RECIST and immune-related response criteria, pharmacokinetics, and adverse events. Exploratory endpoints include secondary efficacy endpoints by PD-LI status, plasma and tumor HA levels, and imaging parameters of tumor blood flow (dynamic contrast-enhanced magnetic resonance imaging [DCE-MRI]) and tumor metabolic activity (positron emission tomography/computed tomography [PET/CT] scans). ClinicalTrials.gov Identifier: NCT02563548. Citation Format: Lyudmila Bazhenova, Philip J. Gold, R. Donald Harvey, Alexander I. Spira, John Nemunaitis, Joaquina C. Baranda, Shirish Gadgeel. A Phase 1b study of PEGPH20 plus pembrolizumab in patients with selected hyaluronan-high solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT032. doi:10.1158/1538-7445.AM2017-CT032

Hyaluronan assessment in tumor microenvironment using new affinity histochemistry assay and scoring method.

e23196 Background: Enzymatic degradation of hyaluronan (HA) by PEGylated recombinant human hyaluronidase PH20 (PEGPH20) induces a remodeling of the tumor microenvironment that impacts tumor behavior and response to therapy. Ventana Medical Systems, Inc., (VMSI) and Halozyme Therapeutics co-developed the VENTANA HA RxDx Assay and a scoring method to evaluate HA in solid tumors to potentially predict which patients might better respond to the addition of PEGPH20 to cancer treatments. Methods: Using Halozyme’s novel recombinant HA binding probe altered to include a rabbit Fc region, VMSI developed a highly specific and sensitive affinity histochemistry assay that binds to HA in tissues. Histological features and staining patterns of 106 samples from Halozyme’s 109-202 Phase 2 study of PEGPH20 combined with nab-paclitaxel plus gemcitabine (AG) vs. AG alone in patients with previously untreated stage IV pancreatic ductal adenocarcinoma (PDA) with clinical outcome data were analyzed to develop a scoring method evaluating HA accumulation. Results: A novel scoring algorithm focused solely on the extracellular matrix (ECM) was developed. An ECM HA staining ≥ 50% of the entire tumor surface at any intensity defines an HA-High PDA. We recommend that a 0.05 cm x 0.2 cm uninterrupted fragment of tumor cells and associated stroma obtained using needle gauges of 16 to 20 G as the minimally required amount of tissue to assess HA in PDA. Based on verification studies performed by VMSI, including an inter-reader precision study with a 94% agreement rate, VMSI scoring method proved to be trainable, reproducible, and transferable to pathologists, supporting an IDE approval for selecting patients in the Phase 3 HALO 109-301 study. Conclusions: Performance of the VENTANA HA RxDx Assay, a robust affinity histochemical assay, has been successfully verified in PDA. Two Halozyme clinical studies in PDA (Phase 2 HALO 109-202 Stage 2 &amp; ongoing Phase 3 HALO 109-301) are expected to validate the VMSI HA scoring method in selecting patients who might better respond to the addition of PEGPH20 to cancer treatments. Current Phase 1/2 studies are exploring the utility of the VENTANA HA RxDx Assay and the pantumor potential of PEGPH20.