Comparison of three different methods to detect bone marrow involvement in patients with neuroblastoma

Patient characteristics

Between July 2009, and April 2015, 81 patients with NB fulfilling the criteria mentioned above were treated at our institution. Sixty-two received anti-GD2 based immune therapy with dinutuximab beta, three patients received salvage chemotherapy and 16 patients had treatment with both dinutuximab beta and chemotherapy. At diagnosis, 72 patients (88.9%) were classified as high-risk and six (7.4%) as intermediate risk according to the INRG classification system. In three patients the INRG system was not applicable due to missing data. Stage 4 NB was diagnosed in 73 (90.1%) and stage 3 NB in eight (9.9%) patients. MYCN amplification was present in 25 (30.9%) and absent in 44 (54.3%) tumors. The MYCN status was unknown in twelve (14.8%) tumors. At the start of the observation period, 18 (22.2%) patients were in complete remission and 63 (77.8%) patients had measurable disease (Table 1).

Table 1 Patient characteristics (N = 81)

For analysis, 369 BM aspirates were obtained and analyzed with CM, FCM and AIPF. Due to the limited number of trephine biopsies, these were excluded from statistical analyses.

The evaluation procedures also included MRI and scintigraphy. In total, 361 mIBG and 354 MRI scans were performed (Table 1).

Overall BM involvement in NB

At the patients´ first BM assessment, NB cells were found in 32/81 (39.5%) aspirates. During the observation period in our institution, four different conditions regarding BM involvement were identified: (i) five patients with initially negative BM who occasionally developed BM involvement; (ii) 44 patients who never presented with NB cells in BM; (iii) 11 patients who were permanently positive for NB cells in BM, and (iv) 21 patients in whom NB cells in BM were absent in some, but not in all assessment time points during treatment (Fig. 1). At the patients´ final assessment, NB BM involvement was proven in 18/78 (23.1%) patients. Sixteen of these 18 patients did not survive. Three patients did not have a final BM assessment.

Fig. 1figure 1

Flow-chart: bone marrow involvement at study entry and during follow-up

Analysis of BM involvement with three different methods

Bone marrow involvement in 369 aspirates was detected by CM in 32 (8.7%) samples, by FCM in 52 (14.1%) samples, and by AIPF in 72 (19.5%) samples.

In each BM aspirate, 400 cells were reviewed by light microscopy. BM samples with reduced cellularity were found in 37.4%. In 22/32 CM positive samples, more than 1% NB cells were detected. The highest single rate of BM infiltration in a sample was 90%. Cytomorphology was reviewed as negative in 91.3%.

By flow cytometry, 2–2.5 × 105 cells were analyzed in each sample. In 52 positive BM samples, the average rate of NB cells was 1.28% (range 0.01–28.3%, median 0.045%).

The mean number of cells analyzed by AIPF was 4.26 × 106 (range 0.22–9.78, median 4.12). In 72 NB positive samples, the average number of analyzed cells was 3.3 × 106 (range 0.22–9.78, median 2.88). The mean NB cell content in the positive samples was 6.29% (range 0.000012–94.5%, median 0.0067%).

Comparison of FCM with AIPF

Results of FCM and AIPF were available at the same time points in 369 BM samples. Neuroblastoma cells were identified in 86 (23.3%) of these aspirates. FCM and AIPF were simultaneously positive in 38/86 (44.2%) samples. In 34 samples, only AIPF revealed a positive result, in 14 samples only FCM.

When NB cells in BM were detected by FCM, according results with AIPF were obtained in 38/52 samples (73.1%). In 12/14 differing results, the degree of BM infiltration was 0.01% or less.

When NB cells in BM were detected by AIPF, according results with FCM were obtained in 38/72 samples (52.8%). In 34 differing results, the degree of BM infiltration was even lower and on average 0.0028% (median 0.00039%).

Cytomorphology compared to FCM and AIPF

With CM, 337/369 (91.3%) of samples were rated negative. Of these 369 samples, AIPF was negative in 297 (80.5%) and FCM in 317 (86%) samples. Positive results with CM were consistently confirmed by AIPF and/or FCM. In addition, AIPF or FCM identified 54 additional cases of sub-microscopic BM infestation in these 337 cytomorphological negative samples.

In 321 samples, results of CM were compared with the results of the immunological methods FCM and AIPF, when these were either both negative or both positive. FCM and AIPF confirmed all 27 CM positive samples and detected eleven additional BM aspirates with NB cells. In those eleven samples, the degree of BM infiltration was low with a median of 0.02% and a mean of 0.11%. In the 27 positive samples, BM infiltration was higher with a median of 0.5%. Cytomorphology was never positive, when FCM and AIPF were negative. These results were statistically significant (Table 2).

Table 2 Comparison of BM assessment methodsAssociation of MRI/mIBG with BM studies

There were 369 assessment time points with either mIBG or MRI in combination with BM aspirates. In 136/443 (36.9%), there was no evidence of NB using all diagnostic tools. In 194 MRI and/or mIBG scans with evidence of disease, CM did not contribute additional information as FCM and/or AIPF confirmed all positive results obtained by CM. Furthermore, NB cells were identified by FCM and AIPF in 11 additional CM negative BM samples. These results were statistically significant (Table 3). MRI and mIBG yielded no evidence of disease in 143 assessments. In 7 of these samples, NB cells were identified in BM either by FCM (4), or by AIPF (3) but not by CM.

Table 3 MRI and mIBG in relation to bone marrow assessment with CM, AIPF, and FCMSurvival

Data on survival were available for 76 patients. Forty-six of 81 (56.8%) patients died of NB, 30 (37%) former study patients are alive with and without residual disease. In five patients from abroad, the current status remains unknown.

All eleven patients with NB cell involvement in the BM during the entire study period did not survive. Nine of nine patients did not survive when NB cells in BM were initially detected by CM. In 23 patients, BM involvement at study entry was only identified by FCM or AIPF. Of these, four (17.4%) are alive, 18 (78.3%) did not survive, and one patient was lost to follow-up.

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