Cartilage boundary lubrication synergism is mediated by hyaluronan concentration and PRG4 concentration and structure

Background Proteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; however, the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. The objectives of this study were to evaluate cartilage boundary lubricating ability of 1) PRG4 + HA in solution at constant HA concentration in a range of PRG4 concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) HA + reduced/alkylated (R/A) PRG4, and 4) hylan G-F 20 + PRG4. Methods Static and kinetic friction coefficients (μstatic,Neq, ) were measured using a previously characterized cartilage-cartilage boundary mode friction test for the following concentrations of purified PRG4 and HA: Test 1: HA (1.5 MDa, 3.3 mg/mL) + PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) + HA (1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) + PRG4 (450 μg/mL). Test 4: HA (3.3 mg/mL) + R/A PRG4 (450 μg/mL). ANOVA was used to compare lubricants within (comparing 6 lubricants of interest) and between (comparing 3 lubricants of interest) test sequences, with Tukey and Fishers post-hoc testing respectively. Results This study demonstrates that both PRG4 and HA concentration, as well as PRG4 disulfide-bonded structure, can alter the cartilage boundary lubricating ability of PRG4 + HA solutions. The boundary lubricating ability of high MW HA + PRG4 solutions was limited by very low concentrations of PRG4. Decreased concentrations of high MW HA also limited the cartilage boundary lubricating ability of HA + PRG4 solutions, with the effect exacerbated by low PRG4 concentrations. The reduction of friction by addition of PRG4 to a cross-linked HA viscosupplement product, but not with addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction where tertiary and quaternary PRG4 structure are important. Conclusions Collectively, these results demonstrate that deficiency of either or both PRG4 and HA, or alterations in PRG4 structure, may be detrimental to SF cartilage boundary lubricating function. This study provides further insight into the nature of cartilage boundary lubrication and advancement towards potential formulation of new intra-articular biotherapeutic treatments for osteoarthritis using PRG4 ± HA.Ye

Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species.

Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.The levels, composition, and MW distribution of all currently known lubricants in SF--HA, lubricin, PLs--vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction

Racial-ethnic variations in phyllodes tumors among a multicenter United States cohort.

BACKGROUND AND OBJECTIVES: Previous studies have identified racial-ethnic differences in the diagnostic patterns and recurrence outcomes of women with phyllodes tumors (PT). However, these studies are generally limited in size and generalizability. We therefore sought to explore racial-ethnic differences in age, tumor size, subtype, and recurrence in a large US cohort of women with PT. METHODS: We performed an 11-institution retrospective review of women with PT from 2007 to 2017. Differences in age at diagnosis, tumor size and subtype, and recurrence-free survival according to race-ethnicity. RESULTS: Women of non-White race or Hispanic ethnicity were younger at the time of diagnosis with phyllodes tumor. Non-Hispanic Other women had a larger proportion of malignant PT. There were no differences in recurrence-free survival in our cohort. CONCLUSIONS: Differences in age, tumor size, and subtype were small. Therefore, the workup of young women with breast masses and the treatment of women with PT should not differ according to race-ethnicity. These conclusions are supported by our finding that there were no differences in recurrence-free survival

Germline Genetic Mutations in a Multi-center Contemporary Cohort of 550 Phyllodes Tumors: An Opportunity for Expanded Multi-gene Panel Testing

BackgroundA paucity of data exists regarding inherited mutations associated with phyllodes tumors (PT); however, some are reported (TP53, BRCA1, and RB1). A PT diagnosis does not meet NCCN criteria for testing, including within Li-Fraumeni Syndrome (TP53). We sought to determine the prevalence of mutations associated with PT.MethodsWe performed an 11-institution review of contemporary (2007-2017) PT practice. We recorded multigenerational family history and personal history of genetic testing. We identified patients meeting NCCN criteria for genetic evaluation. Logistic regression estimated the association of select covariates with likelihood of undergoing genetic testing.ResultsOf 550 PT patients, 59.8% (n = 329) had a close family history of cancer, and 34.0% (n = 112) had ≥ 3 family members affected. Only 6.2% (n = 34) underwent genetic testing, 38.2% (n = 13) of whom had only BRCA1/BRCA2 tested. Of 34 patients tested, 8.8% had a deleterious mutation (1 BRCA1, 2 TP53), and 5.9% had a BRCA2 VUS. Of women who had TP53 testing (N = 21), 9.5% had a mutation. Selection for testing was not associated with age (odds ratio [OR] 1.01, p = 0.55) or PT size (p = 0.12) but was associated with grade (malignant vs. benign: OR 9.17, 95% CI 3.97-21.18) and meeting NCCN criteria (OR 3.43, 95% confidence interval 1.70-6.94). Notably, an additional 86 (15.6%) patients met NCCN criteria but had no genetic testing.ConclusionsVery few women with PT undergo germline testing; however, in those selected for testing, a deleterious mutation was identified in ~ 10%. Multigene testing of a PT cohort would present an opportunity to discover the true incidence of germline mutations in PT patients

Contemporary Multi-Institutional Cohort of 550 Cases of Phyllodes Tumors (2007-2017) Demonstrates a Need for More Individualized Margin Guidelines.

PurposePhyllodes tumors (PTs) are rare breast neoplasms, which have little granular data on margins. Current guidelines recommend ≥ 1 cm margins; however, recent data suggest narrower margins are sufficient, and for benign PT, a negative margin may not be necessary.MethodsWe performed an 11-institution contemporary (2007-2017) review of PT practices. Demographics, surgical, and histopathologic data were captured. Logistic regression was used to estimate the association of select covariates with local recurrence (LR).ResultsOf 550 PT patients, the majority underwent excisional biopsy (55.3%, n = 302/546) or lumpectomy (wide excision) (38.5%, n = 210/546). Median tumor size was 30 mm, 68.9% (n = 379) were benign, 19.6% (n = 108) borderline, and 10.5% (n = 58) malignant. Surgical margins were positive in 42% (n = 231) and negative in 57.3% (n = 311). A second operation was performed in 38.0% (n = 209) of the total cohort, including 51 patients with an initial negative margin (82.4% with < 2 mm), and 157 with an initial positive margin, with residual disease only found in six (2.9%). Notably, 32.0% (n = 74) of those with an initial positive margin did not undergo a second operation, among whom only 2.7% (n = 2) recurred. Recurrence occurred in 3.3% (n = 18) of the total cohort (n = 15 LR, n = 3 distant), at median follow-up of 36.7 months. LR (all PT grades) was not reduced with wider negative margin width (≥ 2 mm v < 2 mm: odds ratio [OR] = 0.39; 95% CI, 0.07 to 2.10; P = .27) or final margin status (positive v negative: OR = 0.96; 95% CI, 0.26 to 3.52; P = .96).ConclusionIn current practice, many patients are managed outside of current guidelines. For the entire cohort, a wider margin width was not associated with a reduced risk of LR. We do not recommend re-excision of a negative margin for benign PT, regardless of margin width, as a progressively wider surgical margin is unlikely to reduce LR

Limited Reporting of Histopathologic Details in a Multi-Institutional Academic Cohort of Phyllodes Tumors: Time for Standardization

BackgroundPhyllodes tumors are rare fibroepithelial neoplasms that are classified by tiered histopathologic features. While there are protocols for the reporting of cancer specimens, no standardized reporting protocol exists for phyllodes.MethodsWe performed an 11-institution contemporary review of phyllodes tumors. Granular histopathologic details were recorded, including the features specifically considered for phyllodes grade classification.ResultsOf 550 patients, median tumor size was 3.0 cm, 68.9% (n = 379) of tumors were benign, 19.6% (n = 108) were borderline, and 10.5% (n = 58) were malignant. All cases reported the final tumor size and grade classification. Complete pathologic reporting of all histopathologic features was present in 15.3% (n = 84) of cases, while an additional 35.6% (n = 196) were missing only one or two features in the report. Individual details regarding the degree of stromal cellularity was not reported in 53.5% (n = 294) of cases, degree of stromal atypia in 58.0% (n = 319) of cases, presence of stromal overgrowth in 56.2% (n = 309) of cases, stromal cell mitoses in 37.5% (n = 206) of cases, and tumor border in 54.2% (n = 298) of cases. The final margin status (negative vs. positive) was omitted in only 0.9% of cases, and the final negative margin width was specifically reported in 73.8% of cases. Reporting of details was similar across all sites.ConclusionIn this academic cohort of phyllodes tumors, one or more histopathologic features were frequently omitted from the pathology report. While all features were considered by the pathologist for grading, this limited reporting reflects a lack of reporting consensus. We recommend that standardized reporting in the form of a synoptic-style cancer protocol be implemented for phyllodes tumors, similar to other rare tumors

Scatterplot of concentrations of hyaluronic acid (HA) by levels of lubricin (A) and phospholipids (PL) (B) and concentration of lubricin by MW distribution of HA (C-F).

<p>HA and lubricin content in SF was determined by ELISA, and ESI-MS/MS was used to quantify phospholipids in 16 control SF, 27 eOA SF, and 22 lOA SF samples as described in Methods. Molecular weight distribution of HA was calculated as the percentage of total HA (= 100%). Linear regression was performed, and Pearson correlation coefficients were calculated.</p

Demographic and disease characteristics of donors.

<p>Inclusion criteria: both genders, age 18–85 years, BMI <40, CRP ≤ 3 mg/L, and all CRP levels for RA. Exclusion criteria: joint infection; severe liver or kidney disease; any surgery within the last 3 months; knee joint surgery within the last 6 months; diabetes mellitus (OA); drug abuse; intra-articular treatment with hyaluronate or corticosteroid treatment within the last 3 months; HIV infection; and tumor/cancer. BMI- body mass index, CRP- C-reactive protein, DAS28- disease activity score 28, K/L-score, Kellgren-Lawrence score, nd, not determined.</p><p>Demographic and disease characteristics of donors.</p

The degree of saturation and length of fatty acids from phosphatidylcholine (PC) and lysophosphatidylcholine (LPC).

<p>Phospholipid species were quantified by ESI-MS/MS in 16 control SF (shaded bars), 27 eOA SF (hatched bars), 22 lOA SF (open bars), and 20 RA SF (grey bars) samples as described in Methods. Subsequently, PC and LPC species were grouped by degree of saturation and number of carbon atoms in the FA chains, respectively, and calculated as a percentage of total PC and LPC, defined 100%. Composition of total FA was determined by gas chromatography for 6 lOA SF samples as described in Methods. Data are presented as median and interquartile ranges <b>(A-D)</b> or mean ± SD <b>(E)</b>. <i>P</i>-values less than 0.05 were considered statistically significant: *0.01< p≤0.05, **0.001A, Relative distribution of PC species according to degree of FA saturation. <b>B,</b> Relative distribution of LPC species by degree of FA saturation. <b>C,</b> Relative distribution of PC species according to chain length of FAs. <b>D,</b> Relative distribution of LPC species by chain length of FAs. <b>E,</b> Degree of saturation and length of fatty acid of total FAs representing 100%.</p