47 Tertiary lymphoid structures (TLS) in desmoplastic melanomas (DM) differ from non-DM-associated TLS by their intratumoral location and enhanced immune activity

BackgroundTertiary lymphoid structures (TLS) are ectopic lymphoid organs that are localized near tumors and other sites of inflammation, and are commonly believed to support antitumor immunity. We previously published studies that show that most desmoplastic melanomas contain TLS, and that TLS in cutaneous metastatic melanomas varied widely in maturation state, in proportions of proliferating T and B cells, and in markers of B cell function, including AID and CD21. Thus, we hypothesized that there may be diversity in TLS function, or immunologic activity, among melanomas. To address this hypothesis, we evaluated TLS in primary desmoplastic melanomas (DM), and non-desmoplastic melanomas (non-DM) for markers of cell proliferation which are indicative of early immune activity.MethodsDM and non-DM tumor specimens, which included primary melanomas (PM), and cutaneous metastatic melanomas (CMM), were evaluated for TLS by multiplex Immunofluorescence histology, by staining for CD20, CD8, PNAd, Ki67, FoxP3, and DAPI. Lymphoid aggregates were identified in 20x spectrally unmixed images by visual inspection and identified as TLS if possessing organized T-cell and B-cell regions in addition to high endothelial venule-like vasculature (PNAd+). TLS were identified in 30 out of 64 screened (48%) CMM, 4/4 non-DM PM, and 8 out of 11 screened (73%) DM. Immune cells localized in TLS were enumerated using Halo software (Indica Labs). Mann-Whitney tests were used for statistical assessments.ResultsDM commonly contain a dense network of fibroblasts and associated stroma, which are not typical for other non-DM (PM and CMM). TLS in DM are located throughout the tumors, intratumorally, in sharp distinction from the peritumoral location of TLS in non-DM. Furthermore, when compared to TLS of non-DM (PM and CMM), TLS of DM contain increased densities of CD20+ B cells (PM p=0.007; CMM p<0.0001) and CD8+ T cells (PM p=0.017; CMM p=0.0006), and a higher proportion of proliferating (Ki67+) CD20+ B cells (PM p=0.04; CMM p=0.009).ConclusionsRecently published studies have identified tumor-associated fibroblasts as the likely initiating cells for TLS formation in murine melanomas. The intratumoral location of TLS in DM puts them in close proximity to the dense fibroblasts and desmoplastic stroma in these tumors, which may be responsible for their intratumoral location. The increased density of B and T cells, and higher proportion of proliferating (Ki67+) B cells, in DM than in non-DM, suggests that there may be greater immune activation, increased germinal center maturation, or less regulation in TLS of DM.Ethics ApprovalApproval was obtained for these studies under IRB protocol #’s 10598 and 19694.

The Impact of Programmed Cell Death on the Formation of Tertiary Lymphoid Structures

Tertiary lymphoid structures are clusters of lymphoid tissue that develop post-natally at sites of chronic inflammation. They have been described in association with infection, autoimmune disorders, cancer, and allograft rejection. In their mature stage, TLS function as ectopic germinal centers, favoring the local production of autoantibodies and cytokines. TLS formation tends to parallel the severity of tissue injury and they are usually indicative of locally active immune responses. The presence of TLS in patients with solid tumors is usually associated with a better prognosis whereas their presence predicts increased maladaptive immunologic activity in patients with autoimmune disorders or allograft transplantation. Recent data highlight a correlation between active cell death and TLS formation and maturation. Our group recently identified apoptotic exosome-like vesicles, released by apoptotic cells, as novel inducers of TLS formation. Here, we review mechanisms of TLS formation and maturation with a specific focus on the emerging importance of tissue injury, programmed cell death and extracellular vesicles in TLS biogenesis.

Evaluation of intra-tumoral (IT) SD-101 and pembrolizumab (Pb) in combination with paclitaxel (P) followed by AC in high-risk HER2-negative (HER2-) stage II/III breast cancer: Results from the I-SPY 2 trial.

508 Background: I-SPY 2 is a multicenter, phase 2 trial using response-adaptive randomization within molecular subtypes defined by receptor status and MammaPrint (MP) risk to evaluate novel agents as neoadjuvant therapy for women with high-risk breast cancer. SD-101 is an investigational Toll-like receptor 9 (TLR9) agonist CpG-C class oligodeoxynucleotide that stimulates the production of IFN-α and interleukin (IL)-12, functional maturation of plasmacytoid dendritic cells, and production of cytotoxic antibodies. IT SD-101 was combined with systemic anti-PD-1 antibody Pb to investigate the antitumor and immunologic activity of this novel immunotherapeutic strategy. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only pts (pts) with HER2- disease were eligible for this treatment. Treatment included weekly P x 12 in combination with IT SD-101 2 mg/ml (1 ml for T2 tumors, 2 ml for >T3 tumors) weekly x 4, then q3 weeks x 2, and IV Pb q3 weeks x 4, followed by doxorubicin/cyclophosphamide (AC) q2-3 weeks x 4 (SD-101+Pembro 4). Pts in the control arm received weekly P x 12 followed by AC q2-3 weeks x 4. The I-SPY 2 methods have been previously published. This investigational arm was eligible for graduation (>85% chance of success in a 300-person phase 3 neoadjuvant trial) in 3 of 10 pre-defined signatures: HER2-, hormone receptor (HR)+/HER2- and HR-/HER2-. Results: 75 pts were randomized and evaluable in SD-101+Pembro 4 treatment arm. The control arm included 329 historical controls enrolled since April 2010. The study arm was stopped due to maximal patient accrual. Pt characteristics were balanced; 56% HR+, 44% HR-. The probability that SD-101+Pembro4 was superior to control exceeded 97% for all eligible tumor signatures, but did not reach the threshold for graduation in any of the signatures. However, it is notable that the rate of pCR/Residual Cancer Burden 1 (RCB1) in the HR+/HER2- signature was 51%. Preliminary safety events for SD-101+Pembro 4 include increased rates of fever, neutropenia, febrile neutropenia, transaminitis, and immune-related events, including adrenal insufficiency. Conclusions: The SD-101+Pembro 4 regimen was active but did not meet the pre-specified threshold for graduation in I-SPY 2. pCR/RCB 1 analysis suggests improved response in the HR+/HER-negative signature compared to control. The clinical significance of these findings needs to be weighed against the potential risk of immune-related toxicities. Clinical trial information: NCT01042379. [Table: see text]

AB0110 DETERMINING THE AMOUNT OF COPPER–CONTAINING PROTEIN, ITS BIOCHEMICAL AND IMMUNOLOGIC ACTIVITY IN RHEUMATOID ARTHRITIS PATIENTS

Background:Production of antibodies to ceruloplasmin (CP) in rheumatoid arthritis is an issue that has not been studied well enough. It was not by chance that this copper–containing alpha 2-glycorpoteid of blood plasma showing multienzymatic properties was chosen as an object of investigation. Data on the content and activity of CP in the blood of rheumatoid arthritis patients are contradictory, which has to do with different approaches to selection of patients and different measuring methods.Objectives:Improving diagnosis of rheumatoid arthritis by determination of antibodies to CP as well as its amount and enzymatic activity.Methods:We studied the serum from 30 apparently healthy individuals, and 108 rheumatoid arthritis patients. Antibodies to CP were determined by enzyme immunoassay using immobilized granulated antigen preparations (modification by Gontar et al, 2002). The amount of CP was determined by enzyme immunoassay according to the method of I.S. Kuzmina et al (1991) using commercial diagnostic agent manufactured by Mechnikov Research Institute for Vaccines and Sera.Results:Enzyme immunoassay showed a mean level of CP antibodies in donor sera of 0,020±0,006 optical density units. The level of normal values of specific antibodies determined as M±2σ included an extinction value in the range 0 – 0,086. The mean value of oxidase activity and the amount of CP in healthy people was 716±26,3 and 921±32 ng/ml, correspondingly. In the process of study we revealed a reliable increase in CP antibody count, the activity and amount of CP in patients with rheumatoid arthritis while in all cases the parameters under study correlated with the degree of disease activity (p<0,05): at activity degree I CP antibodies were 0,098±0,011; CP activity was 954±48,1; CP amount was 1292±73,4. At activity degree II CP antibodies were 0,138±0,007; CP activity was 1163±39,6; CP amount was 1763±69,3. At activity degree III, CP antibodies were 0,182±0,015; CP activity was 1368±89,5; CP amount was 1794±102,8. After a course of hospital treatment was completed, we noted a reliable decrease in the activity and amount of CP (at degree I of rheumatoid arthritis activity p<0,001, at degree II of rheumatoid arthritis activity p<0,01for both parameters; at degree III, p<0,05) compared with baseline findings. A decrease in CP antibodies shows decelerated dynamics, especially in patients with pronounced disease activity, which indicates severe disorders in the immunity that cannot be cured completely within 30 – 40 days of hospital treatment course.Conclusion:Determination of CP antibodies, as well as quantitative content of CP and its oxidase activity can serve as indicators of the activity of rheumatoid arthritis, as well as an accessory criterion of the effectiveness of administered therapy.Disclosure of Interests:None declared

Avoidance of iodine deficiency/excess during pregnancy in Hashimoto’s thyroiditis

Purpose Hashimoto’s thyroiditis is a common disease that also affects pregnant women. We analyzed to what extent the inflammatory process of Hashimoto’s thyroiditis changes with iodine prophylaxis in pregnant women. The target for immunologic activity was levels of thyroid antibodies (TPO). Methods The data were collected retrospectively from 20 consecutive, pregnant patients who had been diagnosed with Hashimoto’s thyroiditis between 01.12.2012 and 01.12.2014 and had received iodine supplementation with 100 µg (n = 1) or 150 µg (n = 19). At least two measurements of TPO antibody levels taken during pregnancy and one after pregnancy were evaluated for each patient in the study group. Results At the end of pregnancy, the average TPO antibody level for the 20 patients studied was 137 IU/ml (+/–214 IU/ml), the lowest being 16 IU/ml and the highest 1000 IU/ml. Despite iodine prophylaxis, levels of TPO antibodies decreased in 18 patients during pregnancy, falling below the reference value of 35 IU/ml in 5 cases. However, in one patient TPO antibodies increased from 60 IU/ml to 237 IU/ml during pregnancy while in another, levels remained constant at 1000 IU/ml. Conclusion Iodine prophylaxis in pregnant women, taken at a dose of 100 or 150 μg daily was shown to have no negative systemic effects on Hashimoto’s thyroiditis in a group of pregnant women with increased TPO levels, and can therefore be recommended for expectant mothers, including those with this disease.

A feasibility study of combined epigenetic and vaccine therapy in advanced colorectal cancer with pharmacodynamic endpoint

Epigenetic therapies may modulate the tumor microenvironment. We evaluated the safety and optimal sequence of combination DNA methyltransferase inhibitor guadecitabine with a granulocyte macrophage-colony-stimulating-factor (GM-CSF) secreting colon cancer (CRC) vaccine (GVAX) using a primary endpoint of change in CD45RO + T cells. 18 patients with advanced CRC enrolled, 11 underwent paired biopsies and were evaluable for the primary endpoint. No significant increase in CD45RO + cells was noted. Grade 3–4 toxicities were expected and manageable. Guadecitabine + GVAX was tolerable but demonstrated no significant immunologic activity in CRC. We report a novel trial design to efficiently evaluate investigational therapies with a primary pharmacodynamic endpoint.Trial registry Clinicaltrials.gov: NCT01966289. Registered 21 October, 2013.

Reactogenicity, safety and immunological efficacy of the live, pentavalent rotavirus vaccine in childhood immunization (results of the multicenter clinical trial)

Introduction. Rotavirus infection (RVI) is the most common cause of severe gastroenteritis in infants and young children worldwide: 600,000 children die annually; it accounts for approximately 3 million hospitalizations and 25 million physician visits each year among children. Preventive vaccination is universally recognized as the most effective measure against this infection.The purpose of the study is assessment of reactogenicity, safety and immunogenicity of the pentavalent live vaccine for RVI prevention in childhood immunization.Materials and methods. The first multicenter prospective, randomized, double-blind, placebo-controlled clinical trial of the pentavalent live vaccine for RVI prevention was conducted in Russia among healthy infants aged 2 months at the time of the first vaccination.Results. The vaccine had a satisfactory safety profile and high immunologic activity when administered in a threedose series for childhood immunization. No negative changes in the children’s health condition were detected during the surveillance monitoring.Discussion. The seroconversion rates, the seroconversion factor and the geometric mean antibody titer were consistent with the results obtained during trials of the above vaccine and its equivalents in other countries.