Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model
<p>Ewing sarcoma (EWS)-FLI1 transcripts detection in ovarian tissue (<span class="html-italic">n</span> = 12). Relative quantification of <span class="html-italic">EWS-FLI1</span> transcripts (B2M reference gene) for the contamination with 0, 10, 100 and 1000 cells. Each symbol represents one ovarian fragment (the average of the duplicates for 1000 cells or triplicates for 10 and 100 cells). The symbol ** means there was a significant difference and <span class="html-italic">p</span> < 0.001.</p> "> Figure 2
<p>EWS-FLI1 transcripts detection in testicular tissue (<span class="html-italic">n</span> = 14). Relative quantification of <span class="html-italic">EWS-FLI1</span> transcripts (B2M reference gene) for the contamination with 0, 10, 100, and 1000 cells. Each symbol represents one testicular fragment (the average of the duplicates for 1000 cells or triplicates for 10 and 100 cells). The symbol ** means there was a significant difference and <span class="html-italic">p</span> < 0.001.</p> "> Figure 3
<p>Sensitivity (SE) and specificity (SP) of detection to distinguish 10 and 100 Ewing cells, and 100 and 1000 Ewing cells in ovarian tissue: (<b>a</b>) The AUC (area under the curve, ROC curve) was 0.94 CI 95% [0.86–1.00] to distinguish 10 and 100 Ewing cells. The optimal decision threshold, determined using Liu and Youden indexes, to distinguish between 10 and 100 EWS cells was 354 EWS-FLI1 transcripts with a sensitivity (SE) of 95% and a specificity (SP) of 86% (in red). For maximal SE (100%) and SP (100%), the cut-offs were 319 and 1150 EWS-FLI1 transcripts, respectively. (<b>b</b>) The area under the curve (AUC) was 0.97 CI 95% [0.92–1.00] between 100 and 1000 Ewing cells. To distinguish between 100 and 1000 EWS cells, the optimal decision threshold determined using Liu and Youden indexes was 3998 EWS-FLI1 transcripts with a SE of 100% and SP of 86% (in red). For a maximal SP (100%), the cut-off was 5528 EWS-FLI1 transcripts.</p> "> Figure 4
<p>Sensitivity and specificity of detection to distinguish 10 and 100 EWS cells, and 100 and 1000 EWS cells in testicular tissue: (<b>a</b>) The AUC was 0.98 CI 95% [0.94–1.00] to characterize 10 and 100 EWS cells. The thresholds to distinguish between 10 and 100 EWS cells were 642 EWS-FLI1 transcripts (Liu and Youden indexes, SE = 92% and SP = 95%) (in red), 521 EWS-FLI1 transcripts for SE = 100% and 749 EWS-FLI1 transcripts for SP = 100%. (<b>b</b>) The AUC was 0.99 CI 95% [0.98–1.00] between 100 and 1000 EWS cells. The cut-offs to distinguish between 100 and 1000 EWS were 3172 EWS-FLI1 transcripts (Liu and Youden indexes, SE = 93% and SP = 100%) (in red) and 2170 EWS-FLI1 transcripts for SE = 100%.</p> "> Figure 5
<p>Illustrations of histology: ovarian tissue (OT) after co-culture with RD-ES cells (at day 7); (<b>a</b>) RD-ES cells (arrow) localized in OT after co-culture stained with hematoxylin and eosin (×10); (<b>b</b>) Area with RD-ES cells disseminated in OT (×20); (<b>c</b>) Area with ovarian follicle (×20).</p> "> Figure 5 Cont.
<p>Illustrations of histology: ovarian tissue (OT) after co-culture with RD-ES cells (at day 7); (<b>a</b>) RD-ES cells (arrow) localized in OT after co-culture stained with hematoxylin and eosin (×10); (<b>b</b>) Area with RD-ES cells disseminated in OT (×20); (<b>c</b>) Area with ovarian follicle (×20).</p> "> Figure 6
<p>Illustrations of histology and immunohistochemistry: RD-ES cells (black arrow) localized in testicular tissue (TT) after co-culture (at day 14) on insert; (<b>a</b>) TT in insert stained with hematoxylin and eosin (×20); (<b>b</b>) ERG-positive staining of RD-ES cells (×20).</p> "> Figure 7
<p>Fluorescence in situ hybridization (FISH) analysis of ovarian tissue using EWSR1 (22q12) dual color break apart rearrangement probe (Vysis) showing RD-ES cells invasion (white arrows) after co-culture (at day 14) (×80). RD-ES cells displayed one fusion (yellow signal), and the simultaneous split pattern of one orange and one green signal (arrows), indicative of a rearrangement of one copy of the EWSR1 gene. The fusion gene is detected by a yellow signal, corresponding to co-localization of the red and green probes.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Experimental Model of EWS Contamination of Germinal Tissues
2.2. Yield of RNA Extraction from Germinal Tissues Frozen Using Slow or Snap Freezing
2.3. Detection of EWS-FLI1 Transcript in Frozen OT and TT Samples Contaminated with RD-ES Cells
2.4. Measurements of Dissemination Potential of RD-ES Cell Lines After Co-Culture with Ovarian and Testicular Samples
2.5. MRD Analysis in Ovarian and Testicular Tissues of Patients with EWS
3. Discussion
4. Materials and Methods
4.1. Patients and Samples
4.2. Freezing and Thawing of Germinal Tissue Samples
4.3. Culture of RD-ES Subclones and Contamination
4.4. Detection of EWS-FLI1 of Type II mRNA Expression by RT-qPCR
4.5. Detection of Potential Dissemination of RD-ES Cells in OT and TT After co-Cultures
4.5.1. OT Co-Culture
Histologic Analysis
Fluorescence in Situ Hybridization (FISH) after Co-Cultures of Ovarian Tissue and RD-ES cells
4.5.2. TT Co-Culture
Immunohistochemistry (IHC)
4.6. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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EWS Patients | Age at Cryopreservation (Years) | Chemotherapy | Metastasis | Outcome |
---|---|---|---|---|
OT A | 16 | Before preservation | Lung | Dead |
OT B | 14 | Before preservation | Lung | Alive |
OT C | 14 | Before preservation | No | Alive |
OT D | 15 | Before preservation | Lung, bone and medullary | Dead |
OT E | 13 | Before preservation | Lung | Alive |
TT A | 7 | Before preservation | Mediastinum | Alive |
TT B | 14 | Before preservation | None | Alive |
TT C | 14 | After preservation | None | Alive |
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Chaput, L.; Grèze, V.; Halle, P.; Radosevic-Robin, N.; Pereira, B.; Véronèse, L.; Lejeune, H.; Durand, P.; Martin, G.; Sanfilippo, S.; et al. Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model. Cancers 2019, 11, 1807. https://doi.org/10.3390/cancers11111807
Chaput L, Grèze V, Halle P, Radosevic-Robin N, Pereira B, Véronèse L, Lejeune H, Durand P, Martin G, Sanfilippo S, et al. Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model. Cancers. 2019; 11(11):1807. https://doi.org/10.3390/cancers11111807
Chicago/Turabian StyleChaput, Laure, Victoria Grèze, Pascale Halle, Nina Radosevic-Robin, Bruno Pereira, Lauren Véronèse, Hervé Lejeune, Philippe Durand, Guillaume Martin, Sandra Sanfilippo, and et al. 2019. "Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model" Cancers 11, no. 11: 1807. https://doi.org/10.3390/cancers11111807