Additional file 3: Table S2. of Inferring clonal structure in HTLV-1-infected individuals: towards bridging the gap between analysis and visualization

Disease status and clonal analysis over time. (PDF 234 kb

Additional file 2: Figure S1. of Inferring clonal structure in HTLV-1-infected individuals: towards bridging the gap between analysis and visualization

Quality control of sequencing output. Figure S2. Trees that can represent hierarchical clonal structures based on clone size. Figure S3. Clonality data for longitudinal samples. Figure S4. Clonality data for cross-sectional samples. (PDF 1247 kb

CSF CXCL10, CXCL9, and Neopterin as Candidate Prognostic Biomarkers for HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

<div><p>Background</p><p>Human T-lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare chronic neuroinflammatory disease. Since the disease course of HAM/TSP varies among patients, there is a dire need for biomarkers capable of predicting the rate of disease progression. However, there have been no studies to date that have compared the prognostic values of multiple potential biomarkers for HAM/TSP.</p><p>Methodology/Principal Findings</p><p>Peripheral blood and cerebrospinal fluid (CSF) samples from HAM/TSP patients and HTLV-1-infected control subjects were obtained and tested retrospectively for several potential biomarkers, including chemokines and other cytokines, and nine optimal candidates were selected based on receiver operating characteristic (ROC) analysis. Next, we evaluated the relationship between these candidates and the rate of disease progression in HAM/TSP patients, beginning with a first cohort of 30 patients (Training Set) and proceeding to a second cohort of 23 patients (Test Set). We defined “deteriorating HAM/TSP” as distinctly worsening function (≥3 grades on Osame's Motor Disability Score (OMDS)) over four years and “stable HAM/TSP” as unchanged or only slightly worsened function (1 grade on OMDS) over four years, and we compared the levels of the candidate biomarkers in patients divided into these two groups. The CSF levels of chemokine (C-X-C motif) ligand 10 (CXCL10), CXCL9, and neopterin were well-correlated with disease progression, better even than HTLV-1 proviral load in PBMCs. Importantly, these results were validated using the Test Set.</p><p>Conclusions/Significance</p><p>As the CSF levels of CXCL10, CXCL9, and neopterin were the most strongly correlated with rate of disease progression, they represent the most viable candidates for HAM/TSP prognostic biomarkers. The identification of effective prognostic biomarkers could lead to earlier detection of high-risk patients, more patient-specific treatment options, and more productive clinical trials.</p></div

Demographics and clinical characteristics of HAM/TSP patients (Training Set).

<p>In the Training set, deteriorating patients were significantly older, experienced disease onset later in life, had been living with the disease for shorter periods, and were more severely disabled (OMDS).</p>*<p>Stable HAM/TSP vs Deteriorating HAM/TSP.</p>**<p>Data are expressed as median [range].</p>†<p>By Mann-Whitney test.</p>‡<p>By Fisher's exact test.</p><p>OMDS = Osame's Motor Disability Score.</p

Demographics and clinical characteristics of HAM/TSP patients (Test Set).

<p>In the Test set, deteriorating patients experienced disease onset later in life and had been living with the disease for shorter periods, but there were no significant differences in current age or OMDS.</p>*<p>Stable HAM/TSP vs Deteriorating HAM/TSP.</p>**<p>Data are expressed as median [range].</p>†<p>By Mann-Whitney test.</p>‡<p>By Fisher's exact test.</p><p>OMDS = Osame's Motor Disability Score.</p

Comparison of potential markers in stable and deteriorating HAM/TSP patients with similar disease durations.

<p>(<b>A</b>) Five CSF marker candidates (CXCL10, CXCL9, neopterin, cell count, and anti-HTLV-1 antibody titer) and four blood marker candidates (proviral load in PBMCs, serum sIL-2R, plasma CXCL9, and plasma CXCL10) were compared among all patients from both the Training and Test Sets pooled together with similar disease durations (range: 7–13 years; no significant difference in duration between stable (n = 8) and deteriorating (n = 10) groups). Data is shown for the top eight CSF markers ranked according to the significance of the difference between the deteriorating and stable subjects. Horizontal bars indicate the median values. The Mann-Whitney <i>U</i>-test was used for statistical analysis. (<b>B</b>) ROC analysis was employed to assess the sensitivities and specificities of the nine markers listed above for discriminating deteriorating HAM/TSP patients from stable patients while controlling for disease duration. AUC = area under the ROC curve; 95% CI = 95% confidence interval.</p

Inclusion and exclusion criteria for this study.

<p>CLEIA = chemiluminescent enzyme immunoassay.</p

Identification of biomarkers associated with clinical progression of HAM/TSP.

<p>(<b>A</b>) Five CSF marker candidates (CXCL10, CXCL9, neopterin, cell count, and anti-HTLV-1 antibody titer) and four blood marker candidates (proviral load in PBMCs, serum sIL-2R, plasma CXCL9, and plasma CXCL10) were compared among a cohort of patients called the Training Set (deteriorating HAM/TSP, n = 11; stable HAM/TSP, n = 14). Data is shown for the top eight CSF markers ranked according to the significance of the difference between the deteriorating and stable subjects. Black circles indicate patients with particularly rapidly progressive HAM/TSP. Horizontal bars indicate the median values. The Mann-Whitney <i>U</i>-test was used for statistical analysis. (<b>B</b>) ROC analysis was employed to assess the sensitivities and specificities of the nine markers listed above for discriminating deteriorating HAM/TSP patients from stable patients. AUC = area under the ROC curve; 95% CI = 95% confidence interval.</p

Selection of candidate biomarkers in the blood by comparing HAM/TSP patients and asymptomatic carriers.

<p>(<b>A</b>) Serum levels of soluble IL-2 receptor (sIL-2R), proviral loads in peripheral blood mononuclear cells (PBMCs), and plasma levels of four chemokines (chemokine (C-X-C motif) ligand (CXCL) 9, CXCL10, CXC11, and chemokine (C-C motif) ligand (CCL) 5) were compared between HAM/TSP patients (HAM; n = 30) and asymptomatic carriers (AC; n = 22). Horizontal bars indicate the median values. The Mann-Whitney <i>U</i>-test was used for statistical analysis. (<b>B</b>) Receiver operating characteristic (ROC) analysis was employed to assess the sensitivities and specificities of the six markers exhibited in part (A) for discriminating HAM/TSP patients from ACs: greater proximity of the ROC curve to the upper left corner indicates higher sensitivity and specificity of the marker. AUC = area under the ROC curve; 95% CI = 95% confidence interval.</p

Selection of candidate biomarkers in the cerebrospinal fluid (CSF) by comparing HAM/TSP patients and control subjects.

<p>(<b>A</b>) CSF levels of total protein, cell count, IgG, neopterin, sIL-2R, and nine chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXC11, CCL17, CCL20, and CCL22) were measured and compared between HAM/TSP patients (HAM; n = 30) and HTLV-1-infected control subjects (control; n = eight: seven ACs and one ATL patient). Data is shown for the top six CSF markers ranked according to the significance of the difference between the HAM/TSP patients and the control subjects. Horizontal bars indicate the median values. The Mann-Whitney <i>U</i>-test was used for statistical analysis. (<b>B</b>) ROC analysis was employed to assess the sensitivities and specificities of the six markers exhibited in part (A) for discriminating HAM/TSP patients from controls. AUC = area under the ROC curve; 95% CI = 95% confidence interval.</p