PMCID string | Title string | Sentences string |
|---|---|---|
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Thus, when comparing AIPF or RT-qPCR-ADRN against the standard-of-care, CM/histology, RT-qPCR-ADRN detected a significantly higher number of patients (p < 0.0001, Mc Nemar’s test), whereas AIPF did not (Table 3). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | The combination AIPF/RT-qPCR-ADRN identified 220/395 positive samples, 124 of which were negative according to CM/histology. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A total of 112 were CM/histology, and sixteen of these were AIPF/RT-qPCR-ADRN. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Accordingly, the combination detected a significantly higher number of patients (p < 0.0001, Mc Nemar’s test) (Table 3). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | When considering discordant pairs the number of samples that tested AIPF/RT-qPCR-ADRN CM/histology was 7.8-times higher as the number of samples that tested positive with CM/histology alone (95% CI, 4.6–13.0) (Table 3). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | The combination also outperformed RT-qPCR-ADRN alone (7.5-times; 95% CI, 4.5–12.6) and AIPF alone (0.9-times; 95% CI 0.6–1.4) (Table 3). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Table 3Comparison of single assay performance versus combined assays against cytomorphology and histology (current standard-of-care)CM & HistologyMc Nemar’s testDiscordant pairsNegativePositiveTotalChi-Squarep-valueRel. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | frequency (range)AIPFNegative239482870.20.70.9 (0.6-1.4)Positive4464108RT-qPCR-ADRNNegative1631617979.5<0.00017.5 (4.5-12.6)Positive12096216RT-qPCR-MESNegative201872880.10.70.9 (0.7-1.3)Positive8225107Combined AIPF & RT-qPCR-ADRNNegative1591617583.3<0.00017.8 (4.6–13.0)Positive12496220Combined AIPF & RT-qPCR-ARDN & RT-qPCR-MESNegative12215137121.1<0.000110.7 (6.3-18.2)Positive16197258 Comparison of single assay performance versus combined assays against cytomorphology and histology (current standard-of-care) Infiltration of DTCs ranged from 1 DTC in 5.30 million bone marrow MNCs to 2.23 million DTCs in 2.48 million MNCs (90% tumor cell content) assessed by AIPF and 0.000007–341% infiltration relative to IMR32 for RT-qPCR-ARDN (Fig. 1E & F). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | In samples with positive results in all three techniques, infiltration (measured by AIPF and RT-qPCR-ADRN) was higher compared to samples positive by only AIPF or RT-qPCR-ADRN, or both. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Disaggregation of data by country showed the same trend in sensitivity and a comparable fraction of positive samples (Supplemental Fig. 1E & F). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Together, these data demonstrate that multi-modal analysis is more effective at detecting bone marrow disease than the current standard of care or each methodology individually. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Furthermore, low tumor cell burden is more frequently detected by RT-qPCR-ADRN alone and, less frequently, by AIPF. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Since bone marrow aspirates were obtained from two sites (left and right iliac crests), we compared results from paired bone marrow samples. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | For the bilaterally tested pairs, 176 AIPF pairs and 175 RT-qPCR-ADRN pairs had identical results for both sides: MRD-negative in 156 AIPF pairs and 121 RT-qPCR-ADRN pairs and MRD-positive in twenty AIPF pairs and 54 RT-qPCR pairs. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | For samples that were positive for both AIPF and RT-qPCR, the majority were positive on both sides (AIPF 57/89, RT-qPCR-ADRN 86/94) (Fig. 2A). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | In contrast, samples that were positive by one technique only, unilateral tumor cell infiltration was more often observed (RT-qPCR-ADRN 61/107, AIPF 3/4) (Fig. 2A). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | The DTC infiltration levels of bilateral positive specimens were significantly higher than the unilateral positive ones, reflecting low infiltration burden in the latter (Fig. 2B). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | However, there was no significant difference between the left and right sides (Supplemental Fig. 2A).Fig. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | 2High tumor cell infiltration at diagnosis is associated with bilateral bone marrow involvement and shift towards unilateral upon therapy-induced reduction of tumor cells. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A AIPF and RT-qPCR positivity on one side only (unilateral) and on both sides (bilateral) in samples where both sides were analyzed for AIPF and RT-qPCR-ADRN (n = 93 AIPF samples positive; n = 201 RT-qPCR-ADRN samples positive). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | B Level of tumor cell infiltration by RT-qPCR-ADRN (left y-axis) and AIPF (right y-axis) according to bilateral (RT-qPCR-ADRN n = 132; AIPF n = 53) and unilateral positive (RT-qPCR-ADRN n = 69; AIPF n = 36) result at all timepoints. ** |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | = 0.0045; ns = not significant High tumor cell infiltration at diagnosis is associated with bilateral bone marrow involvement and shift towards unilateral upon therapy-induced reduction of tumor cells. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A AIPF and RT-qPCR positivity on one side only (unilateral) and on both sides (bilateral) in samples where both sides were analyzed for AIPF and RT-qPCR-ADRN (n = 93 AIPF samples positive; n = 201 RT-qPCR-ADRN samples positive). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | B Level of tumor cell infiltration by RT-qPCR-ADRN (left y-axis) and AIPF (right y-axis) according to bilateral (RT-qPCR-ADRN n = 132; AIPF n = 53) and unilateral positive (RT-qPCR-ADRN n = 69; AIPF n = 36) result at all timepoints. ** |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | = 0.0045; ns = not significant When the standard-of-care MRD diagnostics by CM and histology were included in the analysis, about half of samples positive for all techniques were bilaterally positive by CM. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Whereas those only positive for RT-qPCR-ADRN or CM showed a higher frequency of unilaterally positive cases (Supplemental Fig. 2B). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Interestingly, for two that were one-side-only positive, AIPF and RT-qPCR-ADRN were positive on opposite sides, while CM was negative in these cases. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Comparing different timepoints during the treatment course, the number of bilateral-positive samples decreased from diagnosis, when bilateral positivity was predominant (AIPF bilateral 26/37 (70%); RT-qPCR-ADRN 37/46 (80%)), versus post-induction chemotherapy (AIPF 9/19 (47%); RT-qPCR-ARDN 26/37 (70%)) and end of treatment, when only unilateral MRD was detected (RT-qPCR-ADRN bilateral 0/7) (Supplemental Fig. 2C). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | This shows that bone marrow disease is present at both sides when DTC infiltration is high, i.e. at diagnosis, while in MRD settings, during and end of treatment, more frequently only one side is affected. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Given that multi-modal analysis was more sensitive to detect MRD, we investigated how levels of ADRN mRNAs and immunotherapy targets GD2 and CD56 correlated at different timepoints during high-risk treatment. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Since AIPF can detect absolute neuroblastoma tumor cell counts and RT-qPCR detects neuroblastoma transcripts, we compared the level of calculated infiltration by AIPF with that calculated by RT-qPCR in paired samples. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | As expected, at diagnosis, ADRN mRNAs and the fraction of GD2CD56 cells were high and there was a strong correlation between the bone marrow infiltration of samples positive for AIPF and RT-qPCR (n = 37, Spearman correlation equal to 0.75, 95% CI 0.55–0.87; p < 0.001) (Fig. 3A & Supplemental Fig. 3A). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | During treatment, more bone marrow samples became negative and in those retaining tumor cells, the level of infiltration was reduced. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | At relapse, however, bone marrow disease was again detected by RT-qPCR-ADRN and AIPF (Fig. 3A). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | This kinetics of molecular detection of initial treatment response and subsequent relapse is exemplified by patient 166 (Fig. 3B). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Correlation of infiltration levels was also high when samples during treatment or relapse were considered (n = 395; Spearman correlation 0.69, 95% CI 0.57–0.78; p < 0.001 of samples positive for both techniques (n = 104)) (Fig. 3C). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | When investigating the mRNA markers contributing to MRD detection, most samples were positive for all four ADRN markers: PHOX2B, TH, CHRNA3 and GAP43. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Although PHOX2B had the lowest median expression of all four investigated ADRN markers, it contributed the most as single marker (57/216; 26%) and in combinations (191/216; 88%), while only nine samples were TH or GAP43 single positive and three for CHRNA3 alone (Fig. 3D and Supplemental Fig. 3B). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Especially, when infiltration was low during treatment, PHOX2B remained positive, while other ADRN markers were not expressed (Supplemental Fig. 3C), highlighting high sensitivity of PHOX2B in MRD detection. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Fig. 3Adrenergic RT-qPCR is more sensitive to detect MRD and AIPF detects heterogeneity in immunotherapy targets GD2 and CD56/NCAM. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A Level of tumor cell infiltration according to RT-qPCR-ADRN (left y-axis; given as % relative neuroblastoma cell line IMR32) and AIPF (right y-axis; given as % DTCs detected by AIPF) per timepoint during therapy and progression or relapse (event). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Box plots represent 10–90 percentiles, line shows median. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Heatmap represents number of negative and positive cases. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | B Representative case (patient 166) showing initial response to therapy and relapse. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Bone marrow liquid biopsy assessed by RT-qPCR-ADRN (left y-axis; given as % relative to neuroblastoma cell line IM32) and AIPF (right y-axis; given as % DTCs) per timepoint. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | DE = diagnosis, RE1 = mid-induction chemotherapy, RE2 = end of induction therapy, RE3 = surgery, RE4 = before stem cell transplantation, RE5 = before immunotherapy, RES6 = mid-immunotherapy, RE7 = at end of immunotherapy. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | C Scatter plot showing level of infiltration according to AIPF (y-axis; given as % DTCs) and RT-qPCR-ADRN (x-axis; given as % relative to neuroblastoma cell line IMR32) in bone marrow from all timepoints analyzed by both techniques (n = 395; Spearman correlation = 0.69, 95% CI 0.57–0.78; p < 0.001 of samples positive for both techniques (n = 104)). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | D Adrenergic mRNA-marker (co-)expression by RT-qPCR on samples with positive result all timepoints (n = 215). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | E Automated immunofluorescence plus iFISH (AIPF) of patient 166 showing heterogeneous levels of GD2 and CD56/NCAM at event versus homogeneous GD2 at diagnosis (DE). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Tumor cell identity was confirmed by iFISH for MYCN amplification and NME as reference. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | F Representative images of tumor marker protein expression by imaging mass cytometry (IMC) in patient 166 illustrating loss of GD2 at event (relapse). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | G Imaging mass cytometry (IMC) of patient 166. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Tumor marker protein expression (y-axis) in tumor cells at diagnosis (DE) and event (relapse) Adrenergic RT-qPCR is more sensitive to detect MRD and AIPF detects heterogeneity in immunotherapy targets GD2 and CD56/NCAM. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A Level of tumor cell infiltration according to RT-qPCR-ADRN (left y-axis; given as % relative neuroblastoma cell line IMR32) and AIPF (right y-axis; given as % DTCs detected by AIPF) per timepoint during therapy and progression or relapse (event). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Box plots represent 10–90 percentiles, line shows median. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Heatmap represents number of negative and positive cases. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | B Representative case (patient 166) showing initial response to therapy and relapse. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Bone marrow liquid biopsy assessed by RT-qPCR-ADRN (left y-axis; given as % relative to neuroblastoma cell line IM32) and AIPF (right y-axis; given as % DTCs) per timepoint. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | DE = diagnosis, RE1 = mid-induction chemotherapy, RE2 = end of induction therapy, RE3 = surgery, RE4 = before stem cell transplantation, RE5 = before immunotherapy, RES6 = mid-immunotherapy, RE7 = at end of immunotherapy. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | C Scatter plot showing level of infiltration according to AIPF (y-axis; given as % DTCs) and RT-qPCR-ADRN (x-axis; given as % relative to neuroblastoma cell line IMR32) in bone marrow from all timepoints analyzed by both techniques (n = 395; Spearman correlation = 0.69, 95% CI 0.57–0.78; p < 0.001 of samples positive for both techniques (n = 104)). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | D Adrenergic mRNA-marker (co-)expression by RT-qPCR on samples with positive result all timepoints (n = 215). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | E Automated immunofluorescence plus iFISH (AIPF) of patient 166 showing heterogeneous levels of GD2 and CD56/NCAM at event versus homogeneous GD2 at diagnosis (DE). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Tumor cell identity was confirmed by iFISH for MYCN amplification and NME as reference. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | F Representative images of tumor marker protein expression by imaging mass cytometry (IMC) in patient 166 illustrating loss of GD2 at event (relapse). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | G Imaging mass cytometry (IMC) of patient 166. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Tumor marker protein expression (y-axis) in tumor cells at diagnosis (DE) and event (relapse) We also observed heterogeneity of GD2 and CD56 expression in 5/108 (5%) investigated patients. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | These presented with notably heterogeneous levels of GD2 and/or CD56, or their complete absence, either at diagnosis or after marker expression was lost upon treatment or relapse. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | This is exemplified by patient 166, who initially presented with > 90% tumor cells in the bone marrow and high levels of GD2 and CD56. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | This patient displayed slow molecular response to induction chemotherapy and reached MRD-negative status after the consolidation phase, which was followed by anti-GD2 immunotherapy (dinutuximab beta). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | At relapse, bone marrow DTCs reached 1.59%, but tumor cells were negative for GD2 and the fraction of CD56-expressing cells was lower, while all tumor cells carried the neuroblastoma-typical MYCN oncogene amplification (Fig. 3B & E). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Heterogeneity in tumor marker and immunotherapy target expression was further investigated using a more extensive multiplex imaging approach (imaging mass cytometry). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | This revealed, at diagnosis, next to high levels of GD2 and CD56, high expression of adrenergic neuroblast markers GATA-3, PRPH, ELAVL4 and CD24, intermediate expression of the chromaffin marker CHGA, and bone marrow homing marker CXCR4 and Ki-67, indicating proliferative activity. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Only a small fraction of cells carried neural progenitor-specific SOX10 and S100B, or MES markers CD44 and vimentin. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | In contrast, at relapse, the phenotype of tumor cells shifted drastically, with only CD56, GATA-3 and ELAVL4 expression being retained, albeit with high variance at the single-cell level (Fig. 3F & G). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Together, these data show that AIPF can identify patient tumor cells with heterogeneous expression or absence of immunotherapy targets GD2 and CD56, which might be associated with insufficient response to immunotherapy and relapse. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Thus, AIPF and RT-qPCR are complementary approaches to detect MRD with high sensitivity and characterize tumor cell phenotypes as well as therapeutic targets. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Recent reports suggest that neuroblastoma cells can acquire a MES phenotype in vitro and in vivo . |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | As ADRN markers do not detect MES cells and there is reduced expression of GD2 , we investigated POSTN and PRRX1 mRNA expression as markers for MES cells. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | All 395 bone marrow samples were investigated by RT-qPCR-ADRN, RT-qPCR-MES, AIPF and the standard-of-care CM or histology. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Of these, 273 (69%) were positive by at least one of the techniques (Supplemental Fig. 4A). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Thirty-nine percent (107/273) were positive for RT-qPCR-MES and 37/273 (14%) showed exclusive positivity for MES mRNAs and were negative by the other techniques (Fig. 4A). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Interestingly, when focusing only on the diagnosis timepoint, none of the samples tested positive by RT-qPCR-MES alone (Supplemental Fig. 4B). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | When combining AIPF with both RT-qPCR methods, 258/395 cases (65%) were RT-qPCR-ADRNMESAIPF; 161 (62%) of these were CM/histology. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A total of 112/395 cases (28%) were CM/histology, and fifteen (13%) of these were RT-qPCR-ADRNMESAIPF. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Accordingly, the combination of RT-qPCR-ADRN, -MES and AIPF detected a significantly higher number of samples as positive (p < 0.0001, Mc Nemar’s test) (Table 3).Fig. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | 4Mesenchymal markers identify MRD in adrenergic negative and AIPF negative bone marrow liquid biopsies prior to relapse. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | A Contribution of mesenchymal mRNA RT-qPCR-markers (RT-qPCR-MES),RT-qPCR-ADRN, AIPF and cytomorphology (CM)/histology. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Venn diagram shows samples positive for at least one technology. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Each circle represents positive results of one technique. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | N = 395 samples analyzed by all techniques; N = 273 samples positive by ≥ 1 technique. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | B MES-positive samples by RT-qPCR-MES. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Level of infiltration according to AIPF (y-axis; given as % DTCs) and RT-qPCR-ARDN (x-axis; given as % relative to neuroblastoma cell lines IMR32) (n = 107; Spearman correlation = 0.59, 95% CI 0.33–0.77; p < 0.001 of samples positive for both techniques (n = 38). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | C Level of infiltration by AIPF (y-axis; given as % DTCs) versus MES-positivity (x-axis). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Box plots represent 10–90 percentiles, line shows median. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Heatmap represents number of negative and positive cases. * |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | = 0.0377. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | D Level of infiltration by RT-qPCR-ADRN (y-axis; given as % relative to neuroblastoma cell line IMR32) versus MES-positivity (x-axis). |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Box plots represent 10–90 percentiles, line shows median. |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | Heatmap represents number of negative and positive cases. ** |
PMC12317575 | Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study | = 0.0029. |
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