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 κ (Fleiss) and α (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 ß-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.

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

BACKGROUND: Loss 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. METHODS: Bone 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. RESULTS: Of 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. CONCLUSIONS: Complete or partial lack of GD2 staining on NB cells in bone marrow is more frequent than currently recognized.

Neuroblastoma messenger RNA is frequently detected in bone marrow at diagnosis of localised neuroblastoma patients.

INTRODUCTION: The clinical importance of the detection of neuroblastoma messenger RNA (mRNA) in bone marrow (BM) of localised neuroblastoma patients at diagnosis remains unclear. In this prospective multicentre study, BM samples of a large cohort, were studied using real-time quantitative polymerase chain reaction (qPCR). METHODS: BM samples at diagnosis from 160 patients with localised neuroblastoma were prospectively collected at Dutch and German centres between 2009 and 2013. qPCR was performed using five neuroblastoma specific markers. The association with other biological factors and the prognostic impact of BM positivity and clinical response was assessed. RESULTS: In 58 out of 160 patients neuroblastoma mRNA was detected in BM. In 47 of the 58 positive samples only one marker was found positive. BM positivity was significantly associated with MYCN amplification (p = 0.02) and deletion of chromosome 1p (p = 0.04). In total 31 patients had an event, of which only five patients had progression to stage IV. BM positivity was not associated with an unfavourable outcome. However, the detection of more than one marker was associated with an unfavourable outcome (systemic or local relapse) (event free survival 48% versus 85%; p = 0.03) in the whole cohort and in the observation group. CONCLUSIONS: BM positivity was associated with unfavourable biological factors and might represent more aggressive tumours. Patients with qPCR positive BM should not be upstaged, because of very few systemic events in the cohort. However, for patients with more than one marker positive a more careful follow-up is advisable. These results need to be verified in a very large cohort of localised patients.

Minimal residual disease detection in autologous stem cell grafts from patients with high risk neuroblastoma.

BACKGROUND: The clinical significance of minimal residual disease (MRD) detected by real-time quantitative PCR (qPCR) in autologous stem cell grafts in high risk neuroblastoma is still controversial. In this retrospective multicenter study, autologous stem cell grafts of a large cohort were studied using a panel of RNA markers. PROCEDURE: From 104 patients with high risk neuroblastoma, who received autologous stem cell transplantation as first line treatment, 66 peripheral blood stem cells (PBSC) and 38 CD34+ selected grafts were retrospectively collected at 2 Dutch and 12 German centers between 1997 and 2010. To investigate graft contamination qPCR was performed by using 5 neuroblastoma specific markers (PHOX2B, TH, DDC, CHRNA3, and DBH). RESULTS: In PBSC 6/66 (9%) and in CD34+ selected grafts 3/38 (8%) samples were contaminated. Graft contamination was not associated with an unfavorable outcome (5-years OS, 66% vs. 50.5%; P=0.6 and 5-years EFS, 22% vs. 35%, P=0.7). In multivariate Cox analysis BM MRD at time of harvest was significantly associated with survival (P=0.008 OS and P=0.002 EFS), but graft contamination was still not associated with an unfavorable outcome (P=0.9 OS and P=1 EFS). CONCLUSIONS: Graft contamination is very infrequent in this retrospective cohort of patients with no or minimal BM disease prior to stem cell collection and does not influence outcome in univariate and multivariate analysis. The presence of MRD at time of harvest is a strong outcome predictor. However, these results will have to be verified in a large prospective study.

Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow.

Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.

Lacking immunocytological GD2 expression in neuroblastoma: report of 3 cases.

Immunocytological bone marrow assessment for contamination with neuroblastoma cells is based on their characteristic GD2 surface staining. Neuroblastoma without GD2 expression have been rarely and only after antibody therapy reported. Conventional cytology was performed using Pappenheim staining. For immunocytology, the APAAP method was utilized with the 14G2a anti-GD2 mouse monoclonal antibody. 7 x 10(5) cells on cytospin preparations were investigated. In 2003, 288 bone marrow samples from 191 neuroblastoma patients were investigated by cytology and immunocytology. Three cases demonstrated GD2 negativity on cytologically unambiguous neuroblastoma cells. Two female cases (94 and 37 months of age) with stage 4 neuroblastoma had GD2 expressing neuroblastoma cells in bone marrow at diagnosis. At 2nd relapse 25 and 23 months after diagnosis and 8 months and 12 months after anti-GD2 antibody treatment (ch14.18), the bone marrow infiltrating neuroblastoma cells lacked GD2 staining. The third patient, a 63-month-old girl with bone marrow replacement by neuroblastoma cells showed at diagnosis a mixture of GD2-unstained tumor clumps and very weakly stained neuroblastoma cells. Neuroblastoma cells may lack GD2 expression at diagnosis and at recurrence. This observation has diagnostic and therapeutic implications.