Unraveling heterogeneity within ACPA-negative rheumatoid arthritis: the subgroup of patients with a strong clinical and serological response to initiation of DMARD treatment favor disease resolution

Background Rheumatoid arthritis (RA) is a heterogeneous disease, as evidenced by the differences in long-term outcomes. This applies especially to anti-citrullinated protein antibodies (ACPA)-negative RA, where a proportion achieves sustained DMARD-free remission (SDFR; sustained absence of synovitis after DMARD cessation). Differentiation of RA patients who will achieve SDFR can guide personalized treatment/tapering strategies. Although this subgroup remains scarcely discerned, previous research demonstrated that these RA patients are characterized by an early clinical response (DAS remission after 4 months) after DMARD start. We studied whether, in addition to this clinical response, a specific biomarker response can further distinguish the subgroup of RA patients most likely to achieve SDFR. Methods In 266 RA patients, levels of 12 biomarkers (SAA/CRP/MMP-1/MMP-3/resistin/leptin/IL-6/TNF-R1/YKL-40/EGF/VEGF/VCAM-1), in the first 2 years after diagnosis, were studied in relation to SDFR, stratified for ACPA status. Subsequently, biomarkers associated with SDFR development were combined with early DAS remission to study its additional value in defining subgroups. Since most biomarker levels are not routinely measured in clinical practice, we explored how this subgroup can be clinically recognized. Results ACPA-negative RA patients achieving SDFR were characterized by high baseline levels and stronger decline in MMP-1/MMP-3/SAA/CRP after DMARD-start, respectively 1.30×/1.44×/2.12×/2.24× stronger. This effect was absent in ACPA-positive RA. In ACPA-negative RA, a strong biomarker decline is associated with early DAS remission. The combination of both declines (clinical, biomarker) was present in a subgroup of ACPA-negative RA patients achieving SDFR. This subgroup can be clinically recognized by the combination of high baseline CRP levels (≥ 3 times ULN), and early DAS remission (DAS4 months < 1.6). This latter was replicated in independent ACPA-negative RA patients. Conclusions ACPA-negative RA patients with early DAS remission and a strong biomarker response (or baseline CRP levels ≥ 3× ULN) are most likely to achieve SDFR later on. This could guide personalized decisions on DMARD tapering/cessation in ACPA-negative RA.

Large-scale analysis of longitudinal skin gene expression in systemic sclerosis reveals relationships of immune cell and fibroblast activity with skin thickness and a trend towards normalisation over time

ObjectivesDetermine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time.MethodsA total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions.ResultsGene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up.ConclusionsSkin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design.

Increase in the Complement Activation Product C4d and the Terminal Complement Complex sC5b-9 Is Associated with Disease Severity and a Fatal Outcome in Necrotizing Soft-Tissue Infection

The hyperinflammatory burden is immense in necrotizing soft-tissue infection (NSTI). The complement system is a key during the innate immune response and may be a promising target to reduce the inflammatory response, potentially improving the clinical outcome. However, complement activation and its association to disease severity and survival remain unknown in NSTI. Therefore, we prospectively enrolled patients with NSTI and sampled blood at admission and once daily for the following 3 days. Plasma C4c, C4d, C3bc, and C3dg and the terminal complement complex (TCC) were evaluated using ELISA techniques. In total, 242 patients were included with a median age of 62 years, with a 60% male predominance. All-cause 30-day mortality was 17% (95% confidence interval [CI] 13–23) with a follow-up of &#x3e;98%. C4c and C3dg were negatively correlated with Simplified Acute Physiology Score II (<i>Rho</i> −0.22, <i>p</i> &#x3c; 0.001 and <i>Rho</i> −0.17, <i>p</i> = 0.01). Patients with septic shock (<i>n</i> = 114, 47%) had higher levels of baseline TCC than those in non-shock patients (18 vs. 14, <i>p</i> &#x3c; 0.001). TCC correlated with the Sequential Organ Failure Assessment (SOFA) score (<i>Rho</i> 0.19, <i>p</i> = 0.004). In multivariate Cox regression analysis (adjusted for age, sex, comorbidity, and SOFA score), high baseline C4d (&#x3e;20 ng/mL) and the combination of high C4d and TCC (&#x3e;31 arbitrary units/mL) were associated with increased 30-day mortality (hazard ratio [HR] 3.26, 95% CI 1.56–6.81 and HR 5.12, 95% CI 2.15–12.23, respectively). High levels of both C4d and TCC demonstrated a negative predictive value of 0.87. In conclusion, we found that in patients with NSTI, complement activation correlated with the severity of the disease. High baseline C4d and combination of high C4d and TCC are associated with increased 30-day mortality. Low baseline C4d or TCC indicates a higher probability of survival.

The Stop Signal Task for Measuring Behavioral Inhibition in Mice With Increased Sensitivity and High-Throughput Operation

Ceasing an ongoing motor response requires action cancelation. This is impaired in many pathologies such as attention deficit disorder and schizophrenia. Action cancelation is measured by the stop signal task that estimates how quickly a motor response can be stopped when it is already being executed. Apart from human studies, the stop signal task has been used to investigate neurobiological mechanisms of action cancelation overwhelmingly in rats and only rarely in mice, despite the need for a genetic model approach. Contributing factors to the limited number of mice studies may be the long and laborious training that is necessary and the requirement for a very loud (100 dB) stop signal. We overcame these limitations by employing a fully automated home-cage-based setup. We connected a home-cage to the operant box via a gating mechanism, that allowed individual ID chipped mice to start sessions voluntarily. Furthermore, we added a negative reinforcement consisting of a mild air puff with escape option to the protocol. This specifically improved baseline inhibition to 94% (from 84% with the conventional approach). To measure baseline inhibition the stop is signaled immediately with trial onset thus measuring action restraint rather than action cancelation ability. A high baseline allowed us to measure action cancelation ability with higher sensitivity. Furthermore, our setup allowed us to reduce the intensity of the acoustic stop signal from 100 to 70 dB. We constructed inhibition curves from stop trials with daily adjusted delays to estimate stop signal reaction times (SSRTs). SSRTs (median 88 ms) were lower than reported previously, which we attribute to the observed high baseline inhibition. Our automated training protocol reduced training time by 17% while also promoting minimal experimenter involvement. This sensitive and labor efficient stop signal task procedure should therefore facilitate the investigation of action cancelation pathologies in genetic mouse models.

Circulating lipids and breast cancer prognosis in the Malmö diet and cancer study

Purpose Examine the association between circulating lipids and breast cancer outcomes in patients enrolled in the Malmö Diet and Cancer Study (MDCS). Patients and methods Circulating lipid levels were measured in blood sampled upon enrollment in the female MDCS cohort (N = 17,035). We identified all MDCS participants with incident invasive breast cancer diagnosed between 1991 and 2014. Follow-up time began at breast cancer diagnosis and continued until the first event of breast cancer recurrence, death, emigration, or 5 years of follow-up. We estimated the incidence rates of recurrence at 5 years and fit Cox regression models to compute crude and adjusted hazard ratios (HRs) with 95% confidence intervals (95% CI) of breast cancer recurrence as well as all-cause mortality according to cohort-specific tertiles of apolipoprotein A-1 (Apo A-1) and apolipoprotein B (Apo B). Results We enrolled 850 eligible patients. During the 5 years of follow-up, 90 invasive breast cancer recurrences were diagnosed over 3807 person-years. In multivariable analyses, high baseline levels of Apo B were associated with an increased rate of recurrence (tertile 3 vs. 1, HR = 2.30 [95% CI 1.13–4.68]). However, high baseline levels of Apo B were not associated with all-cause mortality (tertile 3 vs. 1, HR = 1.23 [95% CI 0.68–2.25]). We observed no associations between levels of Apo A-1 and recurrence (tertile 3 vs. 1, HR = 1.34 [95% CI 0.70–2.58]) or all-cause mortality (tertile 3 vs. 1, HR = 1.12 [95% CI 0.61–2.05]). Conclusion High pre-diagnostic levels of Apo B were associated with an increased risk of recurrence among breast cancer patients. Circulating Apo A-1 was not associated with breast cancer outcomes.

887 Repolarization of T cells by AMV564 in patients progressing on checkpoint blockade

BackgroundMDSC produce numerous immune-suppressive factors and are associated with poor outcomes across different cancers. They are frequently elevated in patients experiencing inadequate benefit from checkpoint blockade and there is a crucial need for therapies for this patient population. MDSC are recruited from bone marrow in response to both tumor signaling and T cell activation, and their accumulation in tumors and lymphatics can limit the potential benefits of immunostimulatory therapies. AMV564 is a bivalent T cell engager that selectively depletes MDSC. In a phase 1 study, pharmacodynamic analyses revealed significant depletion of MDSC, T cell activation, expansion of the T cell repertoire and an IFN-gamma-dominant cytokine profile with comparatively limited IL6 induction.1 Monotherapy activity including a confirmed RECIST complete response was observed. The clinical and pharmacodynamic profiles of AMV564 are being further evaluated in specific patient cohorts, including patients progressing on checkpoint blockade.MethodsIn a phase 1b expansion study (NCT04128423), patient cohorts with cancers more likely to include actionable tumor antigens were selected for treatment with AMV564, with most patients representing checkpoint treatment failures. An additional cohort of patients included heterogeneous tumor types stratified by tumor mutation burden (TMB) score from circulating tumor DNA. Pharmacodynamic analyses including direct immunophenotyping (flow cytometry) of T and myeloid cell compartments in peripheral blood were performed on patients treated with AMV564 (15 µg daily for 10 of 21 days by subcutaneous injection).ResultsChanges in myeloid and T cell profiles consistent with the pharmacodynamic signature of AMV564 were observed in patients receiving AMV564 despite one or more prior lines of checkpoint blockade therapy. Notably, both high baseline MDSC and elevated induction of MDSC after T cell activation were apparent (figure 1). Control of MDSC by AMV564 was associated with increases in both effector CD8 and CD4 T cells (figure 2). Extremely elevated levels of regulatory T cells were often observed: after treatment with AMV564, a Th-1-like repolarization of these cells was apparent, often associated with reduction in CD25 (figure 3).Abstract 887 Figure 1Significantly higher induction of M-MDSC is apparent in patients previously receiving checkpoint blockade (CPB) after T cell activation by AMV564.Abstract 887 Figure 2Treatment with AMV564 promotes increases in effector CD8 and CD4 T cells in patients previously treated with CPB (examples shown are Merkel cell carcinoma (MCC) and head and neck squamous cell carcinoma (HNSCC)).Abstract 887 Figure 3Th-1 like repolarization of Treg is apparent in patients previously treated with CPB (MCC, HNSCC examples) after treatment with AMV564 (a). Example CD25 low and T-Bet high cells in HNSCC patient (arrow, b).ConclusionsTreatment with AMV564 yielded substantial reductions in MDSC and favorable polarization of CD8 and CD4 T cells, including Th1-like polarization of Treg. This signature was apparent in patients previously treated with checkpoint inhibitors, despite strong induction of MDSC in response to T cell activation, and high baseline levels (>20%) of Treg.Trial RegistrationNCT04128423ReferencesSmith V, Eckard S, Rettig MP, et al. AMV564, a bivalent, bispecific T-cell engager, depletes myeloid derived suppressor cells and activates T cells in cancer patients. Cancer Res 2020;80(16 Supplement):5699.Ethics ApprovalThis study was approved by the Institutional Review Board (IRB) or Independent Ethics Committee (IEC) at each participating institution (including Ohio State University, MD Anderson Cancer Center, Duke University, University of California Los Angeles, Advent Health, Christ Hospital). All participants gave informed consent for samples used to generate pharmacodynamic data. No sensitive of identifiable information is included.

Rituximab Immunomonitoring Predicts Remission in Membranous Nephropathy

Primary membranous nephropathy (pMN) is an autoimmune kidney disease and a common cause of nephrotic syndrome in adults. Rituximab is becoming a first line therapy for patients with persistent nephrotic syndrome with proven safety and efficacy, achieving remission in 60%–80% of cases. For the remaining 20%–40% of patients there is an urgent need to identify early biomarkers of resistance to rituximab to adapt therapeutic management. In nephrotic patients, rituximab is found in the blood more transiently than in other autoimmune diseases without proteinuria, due to rituximab wasting in the urine. However, rituximab immunomonitoring is not routinely performed. We evaluated the predictive value of serum rituximab levels in patients with pMN three months after rituximab injection (month-3) on clinical remission rates six months (month-6) and 12 months (month-12) after injection and investigated predictive factors for serum rituximab levels at month-3. Sixty-eight patients treated with rituximab between July 2015 and January 2020 from two French nephrology centers were included. We identified residual rituximab levels at month-3 as a novel early predictor of remission at month-6 (p &lt;0.0001) and month-12 (p = 0.001). Reduced likelihood of remission in patients with undetectable rituximab at month-3 was associated with lower serum albumin and higher anti-PLA2R1 titers at baseline and with lower serum albumin, higher proteinuria, higher CD19+ counts and higher anti-PLA2R1 titers during follow-up. In multivariate analysis, high baseline proteinuria and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-6 and high baseline weight and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-12. We identified serum albumin at baseline as a predictive factor for serum rituximab levels at month-3. Patients with serum albumin below 22.5 g/L at baseline had an 8.66-fold higher risk of having undetectable rituximab levels at month-3. Therefore, rituximab immunomonitoring in pMN patients treated with rituximab would allow the detection of patients at risk of treatment failure as early as month-3. Studies are needed to assess whether patients with low residual rituximab levels at month-3 may benefit from an early additional course of rituximab.