Melanotic neuroectodermal tumor of infancy (MNTI) is a rare neoplasm which most commonly arises in the anterior region of the maxilla and always develops during the first year of life (1). It was proposed to be of neural crest origin by Borello and Gorlin (2). Later, Hoshino et al. reported high serum levels of catecholamines in MNTI which suggested a neural crest origin of the tumor (3). The clinical manifestations of MNTI include rapid growth, light blue or pink coloration and displacement of developing teeth. MNTI predominantly affects craniofacial regions in more than 90% cases (4). The largest systematic review of 472 cases showed that 62.2% cases involved maxilla, followed by the skull (15.6%), and mandible (7.8%). MNTI shows a slight male predilection with a 1.27:1 male: female ratio (5). On a computed tomography (CT) scan, MNTI always presents as well-defined hyperdense mass. It shows radiolucent lytic lesion within bone and might have local bone destruction features (6). Although MNTI is generally regarded as a benign tumor, most of the lesions shows aggressive clinical behaviors, including rapidly expanding, destruction of surrounding bony tissues and potential of local recurrence.

Morphologically, MNTI consists of two types of cells with distinct features and different immunohistochemical profile (6). More importantly, only few studies had reported molecular alterations in MNTI, including a case with BRAF V600E mutation (7), and another case with CDKN2A mutation and RPLP1-C19MC gene fusion (8).

Because of the rarity of MNTI, most of the literature have documented single case. We present here with 11 additional cases of MNTI. The clinicopathological characteristics, immunohistochemical profile and molecular alterations, i.e. BRAF V600E mutation, of MNTI are analyzed in this study.

MNTI is an extremely rare neoplasm occurring frequently in male infant younger than 1-year old. Variable names, including melanotic progonoma (10), pigmented congenital epulis (11), retinal anlage tumor (12), etc. have been used for this tumor since 1918. However, all those synonyms are obsolete and not recommended. The name of MNTI is widely accepted now and the tumor is proposed to be of neural crest origin because the tumor produces VMA (2) and has heterogeneous cellular phenotype microscopically which explains by the mesodermal and ectodermal morphological features displayed by neural crest cells at different stages of their ontogeny (13).

Clinically, MNTI may appear as a rapid expansive, lobulated, but non-ulcerated and painless mass. It always presents with light blue or pink appearance (14). In the current study, MNTI mainly affects the maxilla of male infants, which is consistent with the literature (5, 15). Besides the maxilla, other sites, including skull, mandible (16), and noncranial regions, such as brain, epididymis (2), mediastinum, femur and ovary (4, 17) may be affected. Moreau et al. reported 11 cases of MNTI and found that the mean age of the first symptoms and at diagnosis was 2.8 months and 3.2 months, respectively (15). The current study showed similar results, the mean age of the first symptoms and at diagnosis was 3.2 months and 10.6 months, respectively. It should be noticed that there was a time lag between the onset of symptoms and the diagnosis. The reason for this might be that some patients received surgeries at older age because it was not tolerated at a younger age. One limitation for the current study was that none of the cases were subjected to urinary VMA level detection. We could not acquire this information from this cohort. Interestingly, we identified a proportion of cases in this cohort which showed elevated Platelet Count based on the Routine Blood Test. However, it needed more studies and detections before drawing a reliable conclusion.

MNTI is categorized as one type of benign maxillofacial bone and cartilage tumors in 4^th edition of WHO Classification of Head and Neck Tumors. However, radiologically, MNTI always presented with well circumscribed hypodense mass, but may show excessive bone destruction and displacement of dental follicles under CT scan (13). The present case showed a well-defined, unilocular, osteolytic lesion causing expansion and destruction of the maxillary cortical bone, which was in line with the previous publications. The age, tumor location and radiological findings may be suggestive of this tumor. However, we should notice that in the current study, most case were clinically diagnosed with teratoma, cyst or LCH preliminary, only one case were diagnosed as MNTI because this was a recurrent case. Thus, pathological examination is required for a definitive diagnosis.

Late diagnosis might lead to the difficulty in radical resection, because the tumor involves the adjacent structures with rapid invasive growth (4). Once suspected to be MNTI, pathological confirmation might be performed as soon as possible. Histologically, two types of cells with distinct morphological and immunohistochemical phenotype could be seen within this tumor. The tumor cells are arranged in irregular alveolar, tubuloglandular and fissured architecture, with cords and strands intersected by dense fibroblastic stroma. The large melanin-producing polygonal epithelioid cells had vesicular nuclei, prominent nucleoli and eosinophilic cytoplasm, while the small round neuroblast-like cells were surrounded by the epithelioid cells and showed hyperchromatic nuclei and scanty cytoplasm (6). In the present study, the proportion of these two types of cells varied case by case. When a case was predominant with large epithelioid cells (18) or small neuroblast-like cells (19), immunohistochemistry should be performed to confirm the diagnosis because the two type of cells had distinct immunohistochemical phenotype and some of the markers could indicated the presence of the specific type of the cells. In the current study, both large and small cells expressed NSE and Vim, even though that only few small cells expressed Vim. The epithelioid cells were also positive for Pan-CK, HMB45 and the small neuroblast-like cells were positive for Syn. All the tumors were negative for S-100, Melan-A and Des. The immunohistochemical results were in line with the previous publications (6). Those distinct immunohistochemical profile could help distinguishing with other melanin-containing tumors or small round cell tumors, such as melanoma, neuroblastoma, Ewing sarcoma and rhabdomyosarcoma.

The investigations of molecular alterations of MNTI were limited. In 1998, Khoddami et al. (20) studied three typical cases of MNTI and tried to find out the genetic link between MNTI and other small round cell tumors with well-characterized molecular alterations, including MYCN gene amplification, deletion of 1p, the presence of the t(11;22)(q24;q12) and the t(11;22)(p13;q12) translocations. Unfortunately, none of the cases yielded positive results. Melanoma showed a high frequency of BRAF V600E mutation. Gomes et al. (7) performed BRAF V600E mutation detection in three cases of MNTI based on the hypothesis of the similarities with melanoma. They confirmed 1 case with BRAF V600E mutation. Based on those findings, we tried to elucidate the BRAF V600E mutation status in the present cohort. As the pigment can interfere with the fluorescence in the qPCR assay, to ensure the accuracy of the detection, internal and amplification control were included in the ARMS-qPCR experiments. Sanger Sequencing method was used to confirm the results, we also included 2 cases of LCH with confirmed BRAF V600E mutation as positive control. As the results showed, none of the MNTI cases harbored BRAF V600E mutation. The result was inconsistent with Gomes et al.’s study, the tumor enrichment prior to DNA extraction in their investigation might partly explain the mutation detection in one case. However, it had been noticed that another two cases in recent publications were also negative for BRAF V600E mutation (18, 21). Paraffin-embedded samples could influence the molecular analysis, further studies on molecular alterations should be conducted using fresh or frozen samples, if possible. Though all those results were based on limited cases, the frequency of BRAF V600E mutation in MNTI and the individualized treatment plan that might be developed due to this mutation needed more explorations. It had been reported that besides BRAF, other mutated genes, including CDKN2A, NRAS, TP53, NF1 and SF3B1 played significant role in different types of melanoma. The results suggested that those genetic alterations might be also candidates for MNTI, and needed further investigations (22). A better understanding of the molecular biology of MNTI is warranted to improve the efficiency of current treatment strategies, especially for those patients with recurrent or metastatic diseases.

At present, the most common treatment protocol is surgery (16, 23). In the present study, enucleation was the predominant treatment (45.4%), followed by curettage (36.4%), partial resection of maxilla (9.1%) and biopsy (9.1%). Chemotherapy and radiation therapy, either alone or in combination with surgery had also been proposed (5, 24). Because MNTI was consisted of large epithelioid cells and neuroblast-like cells, when it was predominant with neuroblast-like cells, chemotherapy has been suggested as the primary treatment mode (25). Adjuvant chemotherapy was also conducted to avoid recurrence after incomplete surgery (19). Targeted therapy should base on the definitive genetic alterations, which had been inconclusive for MNTI until now.

Unfortunately, the biological behavior of MNTI is hard to be predicted by histopathological features, or immunohistochemical markers. Age of diagnosis (5), gender (4, 5), localization in the maxilla, and R1/R2 resection (4), curettage (16), predominance of a neuroblast-like cell component (26) and Ki-67 index (14) were considered as indicators for high risk of local recurrence. However, those results were controversial because a recent publication reported that none of these factors seemed to be associated with a higher risk of recurrence (15). In the present study, it seemed that only the surgical mode, i.e. curettage showed a correlation with the local recurrence. However, the results in the present study should be considered with caution and a lager sample size of patients were needed to confirm our results and allow statistically relevant conclusions to be drawn.

MNTI, although an extremely rare tumor, it should be added to the differential diagnosis of pediatric population with rapidly growing head and neck lesions. It mainly affects male infants younger than 1-year old with strong preference for maxilla. Distinct histopathological features and immunohistochemical profile are helpful to distinguish from other melanin-containing tumor and small round cell tumors. BRAF V600E mutation in MNTI is very rare and needs further investigations. The factors that contribute to the local recurrence of MNTI are controversial, but the close follow-up for the patients are recommended.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by Ethics Committee of Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine. Written informed consent for participation was not provided by the participants’ legal guardians/next of kin because this is a retrospective study and all the data are anonymous, and the requirement for written consent was therefore waived.

Study conception and design: JL and ZT. Acquisition of data: RHX and CYZ. Analysis and interpretation of data: ZT and JJS. Drafting and revising the article: RHX, LZW, and YHH. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (81872187 and 81702694), Postdoctoral Program of Shanghai Ninth People’s Hospital and the SHIPM-mu fund No. JC201901 from Shanghai Institute of Precision Medicine, Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine and CAMS Innovation Fund for Medical Sciences (CIFMS) (Project No. 2019-I2M-5-037). The funders had no role in study design, data collection, analysis and interpretation, decision to submit the article for publication, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.