Lack of immunocytological GD2 expression on neuroblastoma cells in bone marrow at diagnosis, during treatment, and at recurrence

BackgroundLoss of disialoganglioside 2 (GD2) expression in neuroblastoma (NB) bone marrow cells has been reported in rare cases. This study investigated prospectively the frequency and the patterns of visible GD2 loss at diagnosis, during treatment, and at recurrence. MethodsBone marrow aspirates of patients with new or recurrent stage 4 and 4S NB diagnosed between January 1, 2002 and August 31, 2013 were investigated in parallel by cytology and GD2 immunocytology. Complete negative immunostaining was defined if staining was absent in all and partial if absent in a portion and/or in case of atypical faint staining. ResultsOf 1,261 investigated trial patients of all stages, 474 had unequivocal cytological bone marrow infiltration at initial diagnosis. Thirty-seven patients had tumor cells with complete or partial negative GD2 staining at initial diagnosis, nine during chemotherapy, and 11 at recurrence (altogether 12.0%). The percentage of GD2 negativity in stages 4 and 4S were similar (13% and 9%, respectively). Complete negativity was seen in 14 and partial in 43 cases. Twenty-one cases changed from positive to negative (15 to partial and six to complete) and three cases from negative to positive staining (two to partial and one to complete). The GD2 negative and positive groups were not different regarding tumor sites, molecular characteristics, histology, and tumor markers. Children with stage 4 and GD2 negativity tended to be older at diagnosis (42 vs. 32 months, P = 0.056). Event-free survival and overall survival comparing negative versus positive staining did not show any differences. ConclusionsComplete or partial lack of GD2 staining on NB cells in bone marrow is more frequent than currently recognized

The reliability of bone marrow cytology as response criterion in metastatic neuroblastoma

Background The quantitative assessment of neuroblastoma cell content in bone marrow aspirates for response evaluation has been introduced recently. Data on the concordance of interobserver reports are lacking so far. Methods Investigators of seven European countries representing national reference or large oncological centers convened in 2016. They agreed to quantitatively assess routine bone marrow smears of the participating institutions and to discuss the discrepant results in joint meetings. Results From 2017 through 2019, three cytology rounds with 24, 28, and 28 bone marrow samples were run evaluating the representativity of the smears (yes/[restricted]/no) and the presence of tumor cells (yes/no and %). The comparison of the reports using kappa (Fleiss) and alpha (Krippendorff) statistics demonstrated no robust reliabilities. The agreement on the representativity was moderate to poor, on the presence of tumor cells moderate to good, and on the percentage of tumor cells slight to moderate. Though the value of cytology is unquestioned to detect even tiny metastatic cells in bone marrow, the investigators unanimously agreed that a reliable quantification of the tumor cell content in bone marrow smears is unrealistic. For the key issue of representativity, a new practical definition was developed. Conclusion For any work with bone marrow aspirates, the representativity of the material is of paramount importance. A practical definition is proposed. A reliable quantitative cytological assessment of tumor cell content in bone marrow aspirates is not feasible in metastatic neuroblastoma. Therefore, its use as response criterion should be reconsidered

Hypermethylated RASSF1A as Circulating Tumor DNA Marker for Disease Monitoring in Neuroblastoma

PURPOSE: Circulating tumor DNA (ctDNA) has been used for disease monitoring in several types of cancer. The aim of our study was to investigate whether ctDNA can be used for response monitoring in neuroblastoma. METHODS: One hundred forty-nine plasma samples from 56 patients were analyzed by quantitative polymerase chain reaction (qPCR) for total cell free DNA (cfDNA; albumin and β-actin) and ctDNA (hypermethylated RASSF1A). ctDNA results were compared with mRNA-based minimal residual disease (qPCR) in bone marrow (BM) and blood and clinical patient characteristics. RESULTS: ctDNA was detected at diagnosis in all patients with high-risk and stage M neuroblastoma and in 3 of 7 patients with localized disease. The levels of ctDNA were highest at diagnosis, decreased during induction therapy, and not detected before or after autologous stem-cell transplantation. At relapse, the amount of ctDNA was comparable to levels at diagnosis. There was an association between ctDNA and blood or BM mRNA, with concordant results when tumor burden was high or no tumor was detected. The discrepancies indicated either low-level BM infiltration (ctDNA negative/mRNA positive) or primary tumor/soft tissue lesions with no BM involvement (ctDNA positive/mRNA negative). CONCLUSION: ctDNA can be used for monitoring disease in patients with neuroblastoma. In high-risk patients and all patients with stage M at diagnosis, ctDNA is present. Our data indicate that at low tumor load, testing of both ctDNA and mRNA increases the sensitivity of molecular disease monitoring. It is likely that ctDNA can originate from both primary tumor and metastases and may be of special interest for disease monitoring in patients who experience relapse in other organs than BM