Therapy-naive Patients Research Articles

A2 Phylogenetic investigation of transmitted HIV-1 drug resistance mutations in Denmark, 2009–17

Transmission of HIV-1 resistance mutations among therapy-naïve patients impairs the efficiency of antiretroviral therapy (ART). Therefore, genotypic resistance testing of patients is recommended at baseline, as this both allows for the selection of the correct ART regimen and for surveillance of transmitted drug resistance mutations (TDRM) among therapy naive HIV-1 patients. In Denmark, the occurrence of TDRM in newly diagnosed and therapy naïve HIV-1 patients is monitored through the SERO project. Here, we investigated if the prevalence of TDRM differed between patients within and outside of phylogenetically identified transmission clusters. Samples from 1,227 newly diagnosed HIV-1 patients were sent along with epidemiological information to the Virological Surveillance and Research group at Statens Serum Institut. HIV-1 RNA extraction, RT-PCR and Sanger sequencing of the pol gene was performed using an in-house assay. The sequences were analyzed using BioNumerics v. 6.6 and manually checked for the presence of mixed mutations and analyzed for mutations using the HIVDB 8.4 algorithm implemented at the Stanford database. Sequence alignments were performed in Mafft, and phylogenetic analysis was performed using Mega 6.0 using the Maximum likelihood general time reversible model with 100 bootstrap replicates. Clusters were identified with ClusterPicker at default settings (cluster support = 90%, genetic distance 4.5%). Active clusters contained newly diagnosed patients from the 2015 to 2017 period. HIV-1 sequences from 588 patients belonged to one of 154 clusters, and sequences from 639 patients did not belong to a cluster. Patients in clusters were significantly more likely to be men who have sex with men and subtype B and significantly less likely to be late presenters (Fisher’s test P < 0.05). The TDRM prevalence was significantly higher for patients outside of clusters than within clusters, 16.6 per cent versus 12.1 per cent, respectively (Fisher’s test P < 0.05); however, no significant differences were found in the TDRM prevalence between the 75 active and 79 inactive clusters, nor between small (<3 patients) and large (≥3 patients) clusters. E138A, V179D, and K103N were the three most prevalent TDRMs for both patient groups, whereas M41L differed between them. In Denmark, the TDRM prevalence is lower within clusters than outside, indicating that TDRM cases are either imported and/or belong to yet unidentified clusters.

A Melanoma Patient-Derived Xenograft Model.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Lessons learned: Retrospective assessment of outcomes and management of patients with advanced HIV disease in a semi-urban polyclinic in Epworth, Zimbabwe.

IntroductionHIV continues to be one of the leading causes of infectious death worldwide and presentation with advanced HIV disease is associated with increased morbidity and mortality. Recommendations for the management of advanced HIV disease include prompt screening and treatment of opportunistic infections, rapid initiation of ART and intensified adherence support. We present treatment outcomes of a cohort of patients presenting with advanced HIV disease in a semi-urban Zimbabwean polyclinic.MethodsRetrospective cohort analysis of adult patients enrolled for care at Epworth polyclinic, Zimbabwe between 2007 and end June 2016. Treatment outcomes at 6 and 12 months were recorded. Multivariate logistical regression analysis was undertaken to identify risk factors for presentation with advanced HIV Disease (CD4 count less than 200 cells/mm3 or WHO stage 3 or 4) and risks for attrition at 12 months.Results16,007 anti-retroviral therapy naive adult patients were included in the final analysis, 47.4% of whom presented with advanced HIV disease. Patients presenting with advanced HIV disease had a higher mortality rate at 12 months following enrollment compared to early stage patients (5.11% vs 0.45%). Introduction of a package of differentiated care for patients with a CD4 count of less than 100 cells/mm3 resulted in diagnosis of cryptococcal antigenaemia in 7% of patients and a significant increase in the diagnosis of TB, although there was no significant difference in attrition at 6 or 12 months for these patients compared to those enrolled prior to the introduction of the differentiated care.ConclusionsThe burden of advanced HIV disease remained high over the study period in this semi-urban polyclinic in Zimbabwe. Introduction of a package of differentiated care for those with advanced HIV disease increased the diagnosis of opportunistic infections and represents a model of care which can be replicated in other polyclinics in the resource constrained Zimbabwean context.

Assessing Associations of Synovial Perfusion, Cartilage Quality, and Outcome in Rheumatoid Arthritis Using Dynamic Contrast-enhanced Magnetic Resonance Imaging.

To assess associations of synovial perfusion, cartilage quality, and outcome in rheumatoid arthritis (RA). Synovial perfusion and cartilage quality were assessed by dynamic contrast-enhanced magnetic resonance imaging in metacarpophalangeal joints of 28 treatment-naive patients with RA at baseline and at 3 and 6 months after methotrexate. Analysis was by linear mixed modeling. Synovial perfusion variables were associated with remission (p < 0.05) and cartilage quality (p < 0.004). Maximum synovial enhancement was associated to European League Against Rheumatism response (p < 0.05). Synovial perfusion improved in nonresponders over time (p < 0.05). Synovial perfusion relates to remission, response, and cartilage quality in a cohort of therapy-naive patients with early RA.

Brief Report: Gut Structural Damage: an Ongoing Process in Chronically Untreated HIV Infection.

To investigate the longitudinal changes of gut structural damage in chronically untreated HIV infection. This is a 96-week prospective, single-site, cohort study of antiretroviral therapy-naive HIV-infected participants. Intestinal fatty acid-binding proteins (I-FABP) were used as a surrogate marker of gut structural damage. We assessed changes in I-FABP over 96 weeks and examined the associations between I-FABP, HIV variables, and inflammation. Spearman's correlations and linear mixed-effect models were used to study relationships among variables. A total of 63 HIV-infected, antiretroviral therapy-naive patients were included in this analysis. At baseline, 76% were male; 62% were African American, with median age and body mass index of 40 years and 27 kg/m, respectively. Median HIV-RNA and CD4 T-cell counts were 5520 copies per milliliter and 588 cells per mm, respectively. I-FABP significantly increased from baseline to week 96 (mean change +333.9 pg/mL; P = 0.03), and this increase was associated with viral replication (rho = +0.4; P = 0.03). I-FABP levels were found to be associated with markers of inflammation: sTNFR-II (rho = 0.4, P = 0.02) and sVCAM-1 (rho = 0.04; P < 0.01) at all study time points. Lower baseline CD4 T-cell counts was found to be independently associated with I-FABP progression after adjusting for baseline characteristic variables (P = 0.02). Gut structural damage is an ongoing process in the chronic phase of untreated HIV infection and is largely dependent on viral replication. I-FABP was found to be associated with worse immune function, increased inflammation, and viremia in chronically untreated HIV infection, supporting its role as a biomarker of intestinal barrier dysfunction.

The Impact of 6-month Micronutrient Supplementation on Viral, Immunological, and Mental Health Profile of a Cohort of Highly Active Antiretroviral Therapy-Naive HIV-Positive Patients in Northern Nigeria.

Background:HIV is a chronic disease with inflammatory reactions involving numerous elements of the immune system, resulting in an increased risk for other physical and psychiatric morbidities. Micronutrients, some of which possess anti-inflammatory properties, may help prevent the development of psychological disorders such as anxiety and depression in people living with HIV disease.Objectives:This study examined the profile of viral load, CD4 cell count, C-reactive protein, anxiety, and depression among highly active antiretroviral therapy (HAART)-naive HIV-positive patients receiving micronutrient supplementation over a 6-month period.Materials and Methods:A total of ninety HAART-naïve HIV-infected patients completed the Hospital Anxiety Depression Scale. Their blood samples were taken for serum viral load, CD4 cell count, and C-reactive protein at baseline. They all received a micronutrient supplement for 6 months, and 68 participants who remained in treatment at 6 months were reassessed with the same parameters.Results:After 6 months of micronutrient supplementation, the participants were found to have statistically significantly lower mean scores on the anxiety (t-test = 2.970, P = 0.003) and depression (t-test = 3.843, P = 0.001) subscales. They also had statistically significantly lower median CD4 cell count (P = 0.00) and C-reactive protein serum measures (P = 0.04). The median viral load decreased although the difference was not statistically significant.Conclusion:Micronutrient supplementation may reduce inflammatory reactions, anxiety, and depression in HAART-naive HIV-infected persons.

Modified Nucleosides - Molecular Markers Suitable for Small-volume Cancer?

Modified nucleosides (mNS) in urine are shown to be encouraging markers in cancer, mostly in patients presenting with high tumor mass such is breast and lung cancer. To our knowledge, mNS have not been investigated in head and neck squamous cell carcinoma (HNSCC). HNSCC is characterized by early metastasis into locoregional lymph nodes and slow infiltrating growth, but even in the advanced stage exhibits only a relatively low cancer volume. Therefore, reliable distinction between HNSCC and healthy controls by urinary mNS might pose substantial analytical problems and even more as patients with HNSCC mostly have an increased exposure to tobacco smoke and excessive alcohol consumption which affect the renal mNS pattern. Urinary mNS in samples of 93 therapy-naive patients with HNSCC and 242 healthy controls were quantified by reversed-phase high-performance liquid chromatography. Considering that the circadian rhythm causes diuresis-induced variations in concentration, the mNS-to-creatinine ratio was chosen to compare patients and controls. For sensitivity and specificity in discriminating between patients and controls, the corresponding curve was plotted. Additionally, logistic regression was carried out and a multilayer perceptron neuronal network (NN) was created. Fifteen mNS were detectable in cases and controls; concentrations of 11 were found to be significantly different. The sensitivity and specificity depend on the total volume of the lesion; HNSCC with volume <20 ml was reliably detected, but those with a volume of 20 ml or greater produced amounts of mNS which led to the most accurate detection of HNSCC based on HNSCC-specific mNS patterns. Analysis of urinary mNS allows for detection of small-volume HNSCC, with acceptable specificity and sensitivity if the tumor volume exceeds 20 ml.

Integrase Strand Transfer Inhibitor Resistance Mutations in Antiretroviral Therapy-Naive and Treatment-Experienced HIV Patients in South Korea.

Updated guidelines for the treatment of antiretroviral therapy (ART)-negative patients with HIV recommend integrase strand transfer inhibitor (INSTI)-based regimens. HIV patients are tested for resistance to antiretrovirals, and the reported prevalence of transmitted INSTI resistance remains rare worldwide. However, no data related to INSTI resistance in Korean HIV patients have been reported. We aimed to determine the prevalence of INSTI resistance-related mutations in South Korea. We subjected both ART-naive (n = 58) and ART-experienced Korean HIV patients (n = 41) to genotypic resistance analysis and determined a prevalence of INSTI major resistance mutations of 3.4% (n = 2) among ART-naive patients and 22.0% (n = 9) among ART-experienced patients. In the former group, both major INSTI resistance cases involved the nonpolymorphic E92Q mutation in the integrase strand. Our findings suggest that INSTI resistance testing should be included in the standard resistance screening protocols for Korean HIV patients.

Onset of Retinal Pigment Epithelium Atrophy Subsequent to Anti-VEGF Therapy in Patients with Neovascular Age-Related Macular Degeneration

Purpose: The aim of this study was to evaluate risk factors for the development of retinal pigment epithelium (RPE) atrophy in patients with neovascular age-related macular degeneration (nAMD). Procedures: This post hoc analysis of the prospective RESPONSE study includes 52 therapy-naive nAMD patients without baseline RPE atrophy, who were treated with ≥9 anti-vascular endothelial growth factor (VEGF) injections for ≥3 years. RPE atrophy was assessed via multimodal imaging. Baseline aqueous VEGF and serum complement levels (C3d/C3) were measured. Risk factors for atrophy development were evaluated via logistic regression analysis. Results: Atrophy onset was significantly associated with the duration of nAMD (mean 5.34 years; odds ratio = 1.83, p = 0.012). Anti-VEGF injection number, age, C3d/C3 ratio, baseline intraocular VEGF, or delay to the first treatment had no influence on RPE atrophy. Conclusions: The duration of treatment-requiring nAMD was identified as primary risk factor for the onset of concomitant RPE atrophy after commencing therapy. Targeting concomitant atrophy in nAMD patients might improve the long-term prognosis of the disease.

Three-Dimensional Organoids Reveal Therapy Resistance of Esophageal and Oropharyngeal Squamous Cell Carcinoma Cells.

Background & AimsOropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo. We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas.MethodsWe generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo.ResultsTumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients (P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment.ConclusionsThe single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.

Vitamin D and Bone Mineral Density in HIV Newly Diagnosed Therapy-Naive Patients Without Any Secondary Causes of Osteoporosis.

Bone loss and vitamin D deficiency are common in HIV patients. However, bone health status in newly diagnosed HIV patients has not been thoroughly described. Our aim was to assess the bone mineral density (BMD), bone resorption and vitamin D status in newly diagnosed HIV patients. A prospective observational study in HIV newly diagnosed therapy-naive persons. Patients with secondary causes of osteoporosis were excluded. Bone densitometry (DXA), a bone resorption marker (CTx), 25-hydroxyvitamin D (25OHD), CD4 count and HIV viral load (VL) were done in 70 patients. Vitamin D results were compared with a group of healthy volunteers. All patients were men, mean age 31years (19-50). Low BMD (Z score ≤ 2.0) was found in 13%, all of them in lumbar spine, and in only one patient also in femoral neck. Bone resorption was high in 16%. One out of four participants had low BMD or high bone resorption. Vitamin D deficiency (25OHD < 20ng/mL) was found in 66%. Mean 25OHD in patients was significantly lower than in healthy volunteers (p = 0.04). No associations were found between BMD, CTx, 25OHD and VL or CD4 count. We hypothesize that HIV infection negatively affects bone health based on the results we found among newly diagnosed, therapy-naive, HIV-infected patients, without any known secondary causes of osteoporosis. Low BMD or high bone resorption, are significantly prevalent in these patients. HIV-infected patients had a higher prevalence of vitamin D deficiency than controls, which was not correlated with CD4 count or VL.

Levels of pro-inflammatory biomarkers among HIV patients on highly active antiretroviral therapy (HAART) and HAART naive patients

Inflammation is the immune response of the body to infection and injury. Inflammation aims at eliminating the pathogen or causative agent, removing necrotic cells and tissues and initiating repair process. IL-6. TNF-α and CRP are biomarkers of inflammation. This study sought to determine the pro-inflammatory markers among HIV patients on HAART and HAART naive patients. This was a hospital-based, case control study conducted among 150 participants. HIV patients’ on HAART (n = 50) and HAART naive HIV (n = 50) patients and the control group (n = 50) was HIV Seronegative individuals. Venous blood was drawn from each of the participant. Serial dilutions of standards of TNF-α, IL-6 and CRP were made and mixed with 10 μL of patients’ blood. Absorbance was read at 450 nm within 15 min with Mindray MR-96A reader. Among the study population, IL-6 levels was higher in HAART naive HIV patients 17.66(13.00 - 24.10) ng/L, and these same group also recorded the highest CRP and TNF-α levels of 1453(328.8 – 2510) µg/L and 318.3(123.0 - 497.9) ng/L respectively. The present study revealed elevated levels of pro-inflammatory cytokines (IL-6, CRP and TNF-α) in seropositive HIV groups compared to seronegative group.

Levels of pro-inflammatory biomarkers among HIV patients on highly active antiretroviral therapy (HAART) and HAART naive patients

Levels of pro-inflammatory biomarkers among HIV patients on highly active antiretroviral therapy (HAART) and HAART naive patients

FV9. Nerve ultrasound parameters as biomarkers in CIDP – A prospective 12 months follow-up study

Importance Maintenance or cessation of therapy is an essential question in immune-mediated neuropathies. However, therapeutic biomarkers are of restricted validity, as for example nerve conduction studies (NCS). Ultrasound of the nerves has proven its potential role in the diagnoses of neuropathies; however, its usefulness as a prognostic biomarker is still unclear. Objective To determine whether ultrasound scores and ultrasound morphology are suitable biomarkers for therapeutic response and clinical development. Design Twelve months follow-up observational study. Setting University Hospital Tubingen, Basel and Jena. Participants 79 (including 34 therapy-naive) patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) fulfilling EFNS/PNS-guidelines. Exposures Ultrasound, NCS parameters, and clinical scores (medical research council sum score, MRCSS, and inflammatory neuropathy cause and treatment (INCAT) disability score) at screening visit and after 3, 6, and 12 months. Main outcome Clinical, ultrasound and NCS change over 12 months and its correlation. Results 90% CIDP patients revealed multifocal nerve enlargement as evaluated by the ultrasound pattern sum score (UPSS). The UPSS at baseline did only partially correlate with clinical scores (MRCSS: r 2 = 0.013, p = 0.332; INCAT: r 2 = 0.053, p = 0.048), whereas the NCS data correlated significantly with both ( r 2 = 0.410, and r 2 = 0.443, p r 2 = 0.272 and r 2 = 0.343, p r 2 = 0.054 and r 2 = 0.042, p > 0.25). Furthermore, classifying the ultrasound parameters of nerve, fascicle size and echointensity into three major subtypes, could be shown to be clinically useful biomarkers. Hypoechoic enlarged nerves (class 1), which are found in therapy-naive and chronic CIDP variants (65.5:34.5%), predict the best therapeutic response as an improvement of clincal scores (predictive value up to 0.75). In contrast, enlarged nerves with hyperechoic fascicles or perifascicular tissue (class 2) and those with no or only slighly enlarged nerves (UPSS X 2 -test in favor of class 1: p Conclusion and relevance our results indicate that (i) the quantification of ultrasound signals by the UPSS is a suitable therapeutic biomarker over 12 months, and that (ii) ultrasound-guided nerve morphology may predict therapeutic response.

Increase in transmitted drug resistance in migrants from sub-Saharan Africa diagnosed with HIV-1 in Sweden.

To study the trends of transmitted drug resistance (TDR) in HIV-1 patients newly diagnosed in Sweden, 2010-2016. Register-based study including all antiretroviral therapy-naive patients ≥18 years diagnosed with HIV-1 in Sweden 2010-2016. Patient data and viral pol sequences were extracted from the national InfCareHIV database. TDR was defined as the presence of surveillance drug resistance mutations (SDRMs). A CD4 T-cell decline trajectory model estimated time of infection. Phylogenetic inference was used for cluster analysis. Chi-square tests and logistic regressions were used to investigate relations between TDR, epidemiological and viral factors. One thousand, seven hundred and thirteen pol sequences were analyzed, corresponding to 71% of patients with a new HIV-1 diagnosis (heterosexuals: 53%; MSM: 34%). The overall prevalence of TDR was 7.1% (95% CI 5.8-8.3%). Nonnucleoside reverse transcriptase inhibitor (NNRTI) TDR increased significantly from 1.5% in 2010 to 6.2% in 2016, and was associated to infection and/or origin in sub-Saharan Africa (SSA). An MSM transmission cluster dating back to the 1990s with the M41L SDRM was identified. Twenty-five (1.5%) patients exhibited TDR to tenofovir (TDF; n = 8), emtricitabine/lamivudine (n = 9) or both (n = 8). NNRTI TDR has increased from 2010 to 2016 in HIV-1-infected migrants from SSA diagnosed in Sweden, mirroring the situation in SSA. TDR to tenofovir/emtricitabine, used in preexposure prophylaxis, confirms the clinical and epidemiological need for resistance testing in newly diagnosed patients.

Rheumatoid Arthritis Magnetic Resonance Imaging Score Predicts Therapy Response: Results of the German ArthroMark Cohort.

Markers for treatment response in rheumatoid arthritis (RA) are lacking. The aim of the study was to assess the performance of the RA magnetic resonance imaging (MRI) scoring system (RAMRIS) in combination with serum biomarkers to predict response to methotrexate (MTX) treatment in therapy-naive patients with early RA by using high-field MRI. Twenty-eight patients with RA were prospectively assessed with baseline 3-T MRI of the clinical dominant hand, 3 and 6 months after MTX. The patients met the 2010 American College of Rheumatology/European League Against Rheumatism (EULAR) criteria [average age 56.8 yrs (range 39-74); positive for rheumatoid factor and/or anticyclic citrullinated peptide antibodies; disease duration < 6 mos (range 2-23 weeks)]. RAMRIS and serum biomarkers consisting of various experimental proteins including receptor activator of nuclear factor-κB ligand (RANKL) were obtained. Remission or treatment response was defined according to EULAR. To adjust for intrapersonal correlation, generalized linear mixed models were used. Treatment response at 3 months was associated to low RAMRIS erosion subscores and low total RAMRIS scores (p = 0.019 and 0.03, respectively). Remission at 6 months was associated to low RANKL levels (p = 0.033). In multivariate analyses, response at 3 and 6 months was predicted more accurately with the inclusion of total RAMRIS score, RAMRIS synovitis subscore at the second metacarpophalangeal (MCP) joint, or a combination of the two (p value likelihood ratio test = 0.035, 0.035, and 0.041, respectively). Remission was more accurately predicted with inclusion of RANKL, with no significant predictive effect of MRI. Baseline total RAMRIS can predict EULAR response. RAMRIS synovitis subscore at the second MCP joint and RANKL are associated with response and remission, respectively.

Complex Assessment of Metabolic Effectiveness of Insulin Pump Therapy in Patients with Type 2 Diabetes Beyond HbA1c Reduction.

Background: This prospective single-center study recruited insulin-resistant continuous subcutaneous insulin infusion (CSII) therapy-naive patients with type 2 diabetes (T2D) using insulin analog-based multiple daily injections (MDI) therapy and metformin.Methods: A total of 23 individuals with T2D (70% male), aged a mean ± standard deviation 57.2 ± 8.03 years, with body mass index of 36.2 ± 7.02 kg/m2, diabetes duration of 13.3 ± 4.64 years, and HbA1c of 10.0% ± 1.05% were randomly assigned to a CSII arm or an MDI continuation arm to explore glucose control, weight loss, total daily insulin dose (TDD), and insulin resistance. Insulin dosing was optimized over a 2-month run-in period.Results: At 6 months, patients assigned to the CSII arm achieved a significant mean HbA1c reduction of −0.9% (95% confidence interval [CI] = −1.6, −0.1), while reducing their TDD by −29.8 ± 28.41 U/day (33% of baseline [92.1 ± 20.35 U/day]) and achieving body mass (BM) reduction of −0.8 ± 5.61 kg (0.98% of baseline [104.8 ± 16.15 kg]). MDI patients demonstrated a nonsignificant HbA1c reduction of −0.3% (95% CI = −0.8, 0.1) with a TDD reduction of 5% from baseline (99.0 ± 25.25 U/day to 94.3 ± 21.25 U/day), and a BM reduction of −1.0 ± 2.03 kg (0.99% of baseline [108.9 ± 20.55 kg]). After 6 months, the MDI arm crossed over to CSII therapy. At 12 months, patients continuing CSII demonstrated an additional mean 0.7% HbA1c reduction with 54.6% achieving HbA1c<8%. The final TDD reduction was −9.7 U/day in comparison to baseline; BM increased by 1.1 ± 6.5 kg from baseline. The MDI patients that crossed to CSII showed an HbA1c reduction of −0.5% ± 1.04%, HbA1c response rate of 27.3%, a TDD reduction of −17.4 ± 21.06 U/day, and a BM reduction of −0.3 ± 3.39 kg. Diabetic ketoacidosis or severe hypoglycemia did not occur in either arm.Conclusion: CSII therapy safely and significantly improved metabolic control with less insulin usage, with no sustainable reduction of BM, blood pressure, and lipid profile, in insulin-resistant T2D patients. Treatment adherence and satisfaction in these patients were excellent.

A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate invivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma invivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.

Genotyping of circulating tumor DNA in biliary tract cancer to reveal diagnostic and prognostic information.

291 Background: Biliary tract cancer (BTC) shows increasing incidence and is associated with a high mortality. Diagnosis is difficult due to the frequently occurring inaccessibility of the tumor for biopsy. Noninvasive approaches for (i) assessing and (ii) monitoring the tumor-specific molecular setup are desirable. Characterization of circulating tumor DNA (ctDNA) may help to achieve this goal. Methods: Blood and tumor tissue samples from patients with locally advanced or metastatic BTC prior to and during palliative treatment were collected. Tumor tissue and corresponding ctDNA samples underwent targeted genotyping of the 15 most frequently mutated genes in BTC. Findings were correlated with clinical and imaging data. Results: 24 therapy naive patients with histologically confirmed BTC were included for analyses. The overall mutational concordance (blood/tissue) was 74% and 92% for intrahepatic tumors. The mean variant allele frequency (VAF) in tumor tissue of therapy naïve patients was significantly higher compared to the respective ctDNA (p = 0.0291). In turn, the sequencing depth for ctDNA was significantly deeper (p = 0.0001), enabling us to detect also rare variants. Mean ctDNA VAF at baseline significantly correlated with tumor load (Spearman, r = 0.4073, p = 0.0433) and, exclusively for intrahepatic tumors, also with progression-free survival (Spearman, r = -0.5878, p = 0.0386). During 1st line palliative treatment, we detected a change in the mutational landscape in 36% of cases, Moreover, we had access to ctDNA samples of 5 pretreated patients. While ctDNA samples of therapy naïve patients (n = 23) showed a mean of 0.78 mutations per patient, ctDNA samples of pretreated patients (n = 5) exhibited a mean of 0.4 mutations (p = 0.5519). Conclusions: The molecular landscape of BTC is depicted in ctDNA which may enable to adapt diagnostic and therapeutic strategies to the specific molecular setup present at a certain time. In contrast, the use of targeted resequencing is likely to be insufficient for a comprehensive assessment of treatment induced BTC evolution. For this purpose, we suggest a more extensive analysis of ctDNA by broader sequencing applications and the incorporation of epigenetics.

Evidence of inflammasome activation and formation of monocyte-derived ASC specks in HIV-1 positive patients.

The formation of large intracellular protein aggregates of the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain; also know as PYCARD) is a hallmark of inflammasome activation. ASC speck-forming cells release the highly proinflammatory cytokine IL-1β in addition to ASC specks into the extracellular space during pyroptotic cell death. There ASC specks can propagate inflammation to other nonactivated cells or tissues. HIV-1 retroviral infection triggers inflammasome activation of abortively infected CD4⁺ T cells in secondary lymphatic tissues. However, if pyroptosis occurs in other peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients is currently unknown. We investigated if ASC speck positive cells are present in the circulation of HIV-1-infected patients. PBMCs or plasma of HIV-1 infected, antiretroviral therapy-naive patients were analyzed for the presence of ASC speck⁺ cells or extracellular ASC and compared with healthy controls. Intracellular staining for ASC was employed to detect ASC speck⁺ cells within PBMCs by flow cytometry, and ELISA to detect free ASC in the plasma. ASC multimerization was confirmed by immunoblot. Peripheral blood CD14⁺⁺CD16⁻ monocytes were ASC speck⁺ in HIV patients, but not in healthy controls. In the subgroup analysis, HIV patients with lower CD4⁺ T-cell counts and higher viral load had significantly more ASC speck⁺ monocytes. ASC speck formation did not correlate with Gag expression, coinfection, lactate dehydrogenase or C-reactive protein. Our findings suggest that pyroptotic CD14⁺⁺CD16⁻ classical monocytes of HIV-1-infected patients release ASC specks into the blood stream, a phenomenon that may contribute to HIV-1 induced inflammation and immune activation.