15 research outputs found
Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice
<div><p>Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.</p></div
Cancer family history of the 83 neuroblastoma patients according to p.R337H status.
<p><sup>a</sup> Presence of at least one member of the family with cancer, without criteria for a cancer predisposition syndrome though.</p><p>Cancer family history of the 83 neuroblastoma patients according to p.R337H status.</p
ELISA of plasma Hsp90 levels for earlier engraftment confirmation.
<p>(A) Experimental workflow. Cryopreserved primary ALL cells were thawed and injected into 3 mice for expansion. Fresh xenograph ALL cells were then injected into 6 to 12 secondary animals for experiments. For RS4;11 and TALL-1, cultured cells were directly injected in animals for the experiments. Animals were monitored weekly and sacrificed as indicated. (B) Kinetic of Hsp90 plasma levels over time, following ALL injection. Data points represent mean of 3 animals. The cut-off Hsp90 value (0.1 ng/mL) is indicated. Asterisks represent starting week of sequential sacrifice of animals (Time Point 1). Some groups did not have enough animals for the experiment to be carried out until the third week of sacrifice. (C) Percentage of ALL cells (hCD45+) in bone marrow (BM) and peripheral blood (PB) at the different time points. Data points represent mean of 3 animals. Dotted line represents the usual cut-off (0.5%) for ALL detection by flow cytometry in blood samples. Different ALL cells are represented by colors. Circles; BCP-ALL. Triangles, T-ALL.</p
Literature review on <i>TP53</i> mutations in association with neuroblastoma.
<p>NB–neuroblastoma; LFS–Li-Fraumeni Syndrome</p><p><sup>a</sup> in two cases NB were concomitant with ACT</p><p><sup>b</sup> of these 461 carriers, 11 developed adrenocortical tumors, 2 choroid plexus tumors, 1 glioblastoma multiforme, 1 Burkitt lymphoma and 1 neuroblastoma</p><p><sup>c</sup> All 148 cancer cases were diagnosed in carriers of <i>TP53</i> germline mutations</p><p><sup>d</sup> There is no information on the total number of individuals (carriers) analyzed.</p><p>Literature review on <i>TP53</i> mutations in association with neuroblastoma.</p
Occurrence of Neuroblastoma among <i>TP53</i> p.R337H Carriers
<div><p>The high incidence of adrenocortical tumors and choroid plexus carcinoma in children from South and Southeastern regions of Brazil is associated with the germline p.R337H mutation of <i>TP53</i> gene. The concomitant occurrence of neuroblastoma and adrenocortical tumors in pediatric patients harboring the p.R337H mutation at our institution prompted us to investigate the putative association between p.R337H and pediatric neuroblastoma. Genomic DNA samples from 83 neuroblastoma patients referred to a single institution during the period of 2000–2014 were screened for the p.R337H mutation. Available samples from carriers were investigated for both nuclear p53 accumulation and loss of heterozigosity in tumor. Clinical data were obtained from medical records in order to assess the impact of 337H allele on manifestation of the disease. Seven out 83 neuroblastoma patients (8.4%) were carriers of the <i>TP53</i> p.R337H mutation in our cohort. Immunohistochemical analysis of p.R337H-positive tumors revealed nuclear p53 accumulation. Loss of heterozigosity was not found among available samples. The presence of 337H allele was associated with increased proportion of stage I tumors. Our data indicate that in addition to adrenocortical tumors, choroid plexus carcinoma, breast cancer and osteosarcoma, genetic counseling and clinical surveillance should consider neuroblastoma as a potential neoplasia affecting p.R337H carriers.</p></div
Correlation between plasma Hsp90 level and percentage of ALL cells in the different tissues analyzed.
<p>One representative case of three BCP-ALL or T-ALL analyzed is shown. For complete data refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.s002" target="_blank">S2 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.s003" target="_blank">S3 Fig</a> ELISA Hsp90 and flow cytometry hCD45+ data were transformed to log10 and analyzed by Pearson’s correlation. Correlations between ALL in peripheral blood and in the different tissues are shown for comparisons. Dotted line represents cut-off values for ALL detection by flow cytometry (0.5%) or Hsp90 levels (0.1 ng/mL). Data points correspond to individual samples. Numbers near each data point represent time point of sampling (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.g002" target="_blank">Fig 2</a>). PB; peripheral blood. BM; bone marrow. Circles, BCP-ALL. Triangles, T-ALL.</p
Analysis of three potential leukemia plasma biomarkers.
<p>(A) Peripheral blood B2M, IGFBP-2 and Hsp90 levels (ELISA) in NOD/SCID mice transplanted with a primary human T-ALL or healthy controls. Animals were divided into groups according to the percentage of ALL cells (hCD45+) in peripheral blood by FACS. Data points correspond to individual samples, and horizontal bars correspond to median. *P<0.05; Mann-Whitney U test. (B) Correlation between Hsp90 levels and % ALL cells in peripheral blood. Data points correspond to individual samples. Data were transformed to log10 and analyzed by Pearson’s correlation. (C) Cut-off value of Hsp90 (dotted line) as determined by adding 2 times SD to mean. Animals transplanted with different BCP-ALL (n = 3) and T-ALL (n = 2), sacrificed at the earliest time point (time point 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.g002" target="_blank">Fig 2</a>) were included in the analysis. Leukemia engraftment was confirmed by FACS analysis. Data points correspond to individual samples. PB, peripheral blood; SD, standard deviation.</p
Immunohistochemical p53 staining on p.R337H positive NB tumors.
<p>A, B) Representative images of nuclear p53 accumulation on neuroblastoma cells from patients 3 and 7, respectively (100x magnification).</p
Chemotherapy effects on biomarkers levels.
<p>Plasma biomarkers levels (ELISA) and matched percentage of ALL cells (hCD45+) in peripheral blood (PB) from animals under chemotherapy treatment. Samples were collected for analysis at the beginning (C1 = day 5), middle (C2 = day 19) and end (C3 = day 33) of treatment. Colored bars represent mean ± SD of biomarker levels (left Y axis) for individual mice samples analyzed in duplicate. Different colors represent different treatments. The PI3K inhibitor used was AS605240. Black bars represent percentages of ALL cells in peripheral blood (right Y axis). The dotted line in the Hsp90 graphic represents the ELISA cut-off value. Red arrows highlight lower than expected ELISA values, when compared to levels found in untreated animals with similar percentage of ALL cells.</p
Characteristics of p.R337H positive patients.
<p>NB–Neuroblastoma; ACT–adrenocortical tumor; NA–material not available for analysis; IHC–immunohistochemistry; DBD–DNA-binding domain. Immunohistochemistry was scored as 0 (no cells positive), + (up to 25% of cells positive), ++ (26% to 50% of cells positive), +++ (51% to 75% of cells positive), or ++++ (>75% of cells positive).</p><p><sup>a</sup> Only the mutant allele was found in this tumor. It was not possible to distinguish between homozygosity or loss of heterozygosity with retention of the mutated allele in the tumor.</p><p>Characteristics of p.R337H positive patients.</p