Material and Method: The study included 61 cases with archival bone marrow biopsy tissues. The cases were evaluated regarding the percentage of metastatic tissue and its differentiation. Primary tumor slides were also reviewed to perform the Shimada classification based on the differentiation status and mitosis-karyorrhexis index. The patients’ age, gender, NMYC amplification, clinical risk group, and disease outcome were also noted.

Results: Of the 61 cases, 17 had BM involvement. Of those, eight cases (47.1%) were refractory NB showing disease relapse. Based on BM examination, five cases (29.4%) were categorized as complete response, seven (41.2%) as progressive disease, three (17.6%) as minimal disease, and two (11.8%) as stable disease. The progressive disease category was significantly related with refractory disease and NMYC amplification along with the high-risk category (p =0.002 and p= 0.003 respectively). Undifferentiated histology and presence of more than 20% of tumor tissue in the BM biopsy at diagnosis were significantly associated with the progressive disease category (p=0.01 and p<0.001, respectively).

Conclusion: We conclude that evaluating the percentage of metastatic tumor tissue and tumor differentiation in BM biopsies is of clinical importance in the management of neuroblastoma patients.

In the past few decades, a significant improvement has been observed in the prognosis of certain well-defined NB patient subsets, whereas only a modest improvement has been reported in the prognosis of children of the highrisk groups ¹⁵. The presence of tumor tissue in the BM corresponds to not only advanced stage but also a high-risk category. NB patients with bone marrow involvement carry a high risk of refractory disease and are likely to show a poor prognosis. Therefore BM aspiration and biopsy are quick, easy and cost-effective methods and have become routine standard procedures to evaluate the prognosis in newly diagnosed patients and assess disease response without having to wait for greater tumor burden to develop ^1,10. The information achieved by bilateral trephine BM biopsy interpretation is also valuable for the clinical follow-up strategy ¹. We aimed in the present study to address the clinical importance of BM involvement in the management of NB patients, and also to test the significance of established histopathological criteria in predicting the prognosis.

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Table I: The categorization of response status based on bone marrow involvement.

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Figure 1: Neuroblastoma metastasis in bone marrow biopsy (involvement ratio: 15%). H&E, A x40, B x100, C x400, D: synaptophysin immunohistochemistry x400.

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Figure 2: Neuroblastoma infiltration in bone marrow biopsy (involvement ratio: 70%). H & E; A x40, B x100, C x400, D: synaptophysin immunohistochemistry, x400.

Archival histological slides of the primary tumor tissues of 17 patients were also reviewed by two pathologists (EO, SE) to assess the Shimada category based on tumor differentiation and MKI. Demographic data such as patients’ age and gender, clinical data including tumor NMYC amplification status, stage, patient risk group and refractory status were obtained from the patient records.

Clinical and demographic characteristics of the patients were defined by using descriptive statistics. The statistical relationship of both BM metastatic tumor percentage category and differentiation with clinical and demographic characteristics was evaluated with the chi-square test. A probability level of 0.05 or less was chosen to represent statistical significance. All p-values were two-sided and denoted by p. Fisher’s exact test was used to calculate p values, as the cell frequencies were too small for the standard chi-squared test to be accurate.

Approval for the study was granted by the Local Medical Ethics Committee (approval No. 2021/5-2). All study procedures were performed according to the Declaration of Helsinki principles.

Histologically, four tumors were undifferentiated NBs, and 13 were poorly differentiated. Four cases had low, one case had moderate MKI and 12 cases had high MKI. All tumors had unfavorable Shimada histology. Of the tumors with BM involvement, five (29.4%) were undifferentiated NBs and 12 (73.6%) were poorly differentiated. There were eight cases (47.1%) with BM involvement less than 5%, three (17.6%) with 5-20%, and six (35.3%) with more than 20%. Based on BM involvement criteria, seven cases (41.2%) were progressive disease, five (29.4%) complete response, three (17.6%) minimal disease, and two (11.8%) stable disease.

Table II demonstrates the statistical relationship between BM disease status and the prognostic parameters. There was no statistically significant relationship between disease status and gender, age, tumor differentiation and MKI. A statistically significant relationship was found between progressive disease and BM involvement percentage more than 20% (p<0.001), lack of differentiation of the tumor (p=0.001), relapse (p=0.002), high-risk category (p=0.003), and N-MYC amplification (p=0.003). Progressive disease and NMYC amplification were significantly related with refractory disease (p=0.002 and p=0.003 respectively).

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Table II: The relation between bone marrow status and clinicopathological characteristics of the study group.

Almost half of NBs have metastases at the time of diagnosis ^3,17. Although the most common localization of metastases is the BM, metastases can be observed in the skeleton, lymph nodes, liver, and intracranial and orbital regions ^{3,4,6,7,17,21,22}. Despite aggressive multimodality therapy, the long-term survival rate at diagnosis of metastatic disease for patients older than 18 months is still less than 30% ¹⁵.

BM is the major site of metastasis in advanced neuroblastoma, and detection of even minimal residual neuroblastoma cells in this region is associated with a poor prognosis ^9,17. However, the potential for metastatic spread may not be equal among all patients with NB ¹⁹. Although it is a well-known tumor with its different behaviors and the connections between these behaviors and molecular patterns, the metastatic propagation mechanisms of NBs are not fully understood. Studies have shown that some tumors have a higher risk of metastatic spread than others as a result of searching the factors associated with metastatic spread and BM infiltration ¹⁹.

Since the determination of BM metastasis is a key point in evaluating the course of NB, core biopsies are frequently taken as part of routine bone marrow sampling in pediatric patients in many institutions. An advantage of this sampling is that it allows the use of immunohistochemistry on these samples for detection of tumor cells ¹⁷. Previous studies have shown that immunohistochemical analysis is superior to routine H&E assessment ¹⁷ and it is recommended to use at least three immunohistochemical markers such as synaptophysin, chromogranin, PGP 9.5 and cyclin-D1 to demonstrate tumor tissue ^1,13,17. Moreover, neuroblastic differentiation and the percentage of tumor tissue should be stated in the pathology report ¹.

Amplification of the MYCN gene is observed in approximately 20-35% of all NBs and is an important molecular marker for identifying high-risk patients ^3,4,14. The combination of MYCN overexpression and caspase-8 deletion significantly increases BM metastasis ^3,4. However, it has been reported that BM metastasis is associated with poor prognosis in patients older than 1 year, regardless of MYCN amplification ^10,13. In addition, BM status beyond other known predictive factors such as MYCN copy number was shown to provide independent prognostic information for patients over the age of 1 and with stage 4 NBs ¹⁸. In a study evaluating the MYCN status by using FISH and amplification, BM involvement was reported in 6% of the cases. In this study, all cases were high-risk and stage 4, and the criterion for positive BM involvement was the presence of neuroblastoma in at least 20% of the biopsy ¹⁴.

In the present study, the majority of the cases were in the high-risk group older than 18 months. MYCN amplification was observed in 70% of the cases with BM metastasis. In a study by Jo et al. ^8, similar results were reported. Kuroda et al. ¹³ investigated the presence of circulating tumor cells (CTC) in the peripheral blood and/ or BM micrometastases during treatment in advanced stage NB cases receiving chemotherapy. MYCN amplification was detected in 12 of these cases. However, neither CTC nor persistent BM micrometastases were associated with MYCN amplification. In our study, a statistically significant relationship between progressive disease based on BM involvement and NMYC amplification was found.

In our study, BM involvement over 20% and undifferentiated histology were related to refractory disease. In a study by Russell et al. ^19, low-stage NB was unlikely to have metastatic disease in the BM, and therefore they recommended further investigation of the genetic factors such as MYCN amplification and chromosome 1p deletion in order to better predict which tumors are at risk of metastatic spread. In contrast, the presence of permanent BM micrometastasis after chemotherapy was reported as a predictor of poor prognosis ¹³. In addition MYCN amplification should be evaluated both in the primary tumor and BM metastases although the tumor may show genetic heterogeneity. It should be kept in mind that genetic differences that can be found in the primary tumor, and the metastatic tumor may also affect the treatment process ²³.

In the study by Tian et al. ^10, the presence of BM metastasis was seen in 75% of the cases at the time of diagnosis and 45% of the cases after treatment. Five-year event-free survival was found to be statistically different in patients with residual metastatic disease compared to patients without ¹⁵. In our study, a statistically significant relationship was found between the presence of more than 20% of tumor tissue in the BM biopsy at diagnosis and progressive disease. In conclusion, BM metastasis status at diagnosis and during follow-up appears to be a significant prognostic factor apart along with poor prognostic factors. The crucial cut-off point for BM involvement is 20% and a value over this ratio is significantly related to refractory disease. Our study demonstrates the prognostic and predictive significance of determining the tumor percentage and differentiation of NBs in the BM biopsies at diagnosis and during follow-up.

CONFLICT of INTEREST
The authors have no conflicts of interest to declare.

FUNDING
The authors have declared that they did not receive any financial support for this study.

AUTHORSHIP CONTRIBUTIONS
Concept: SE, EO, Design: SE, EO, Data collection or processing: SE, DI, NO, EO, Analysis or Interpretation: SE, DI, NO, EO, Literature search: SE, EO, Writing: SE, EO, Approval: SE, DI, NO, EO.

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