Rhabdomyosarcoma (RMS) is a rare malignancy, but has a high incidence in pediatric solid tumors, with clinical subtypes such as Embryonal Rhabdomyosarcoma (ERMS), Alveolar Rhabdomyosarcoma ( rhabdomyosarcoma (ARMS), and a rare adult variant of rhabdomyosarcoma (pleomorphic rhabdomyosarcoma, PRMS) [1,2]. However, these tumors have been increasing in recent years with the increasing incidence of malignant tumors. Embryonal rhabdomyosarcoma is the most common subtype of soft tissue tumors in pediatric rhabdomyosarcoma, with a peak age of onset within 10 years and a median age of 5. RMS originates from primitive mesenchymal cells or embryonic muscle tissue and can occur in various parts of the body. In view of the fact that RMS is a highly malignant soft tissue tumor with rapid growth, it is easy to be missed or misdiagnosed clinically, and the overall prognosis is poor due to various clinical treatments, we would like to review the relevant research progress.
1.Molecular biological characteristics of RMS
Specific chromosomal translocations can be detected in 80-85% of ARMS by cytogenetic or molecular biology studies: ‘t(2;13)(q35;q14) or t(1;13)(p36;q14). t(2;13) is found in about 70% of ARMS patients, t(1;13) is seen in 10 -15 % of ARMS patients [3]. The two translocations form the corresponding fusion genes PAX3-FKHR (also known as PAX3-FOXO1) or PAX7-FKHR (also known as PAX7-FOXO1), respectively [4].The proteins encoded by PAX3 and PAX7 play an important role in embryonic primitive myogenesis.The PAX3/PAX7-FKHR fusion protein is a stronger than PAX3/ PAX-FKHR fusion protein is a highly efficient tumor-specific transcription factor with higher expression levels than their corresponding wild-type proteins. high expression of PAX-FKHR induces oncogenic transformation of chicken embryonic fibroblasts and murine NIH3T3 cells. In conclusion, PAX CFKHR is a pleiotropic fusion protein that stimulates cell proliferation, inhibits apoptosis, induces angiogenesis, activates the myelopoietic program and simultaneously inhibits terminal differentiation.PAX3/PAX7-FKHR fusion protein expression is associated with poor prognosis and is often seen in patients with metastases (some presenting with bone marrow metastases) [2].ERMS chromosomal translocations are rarely seen in ARMS. ARMS. a study of a large sample of RMS suggested the existence of only two molecular subclasses of RMS, one being fusion gene positive ARMS and the other including ERMS, fusion gene negative ARMS and other fusion gene negative RMS [4]. One study found the presence of PAX3-NCOA1 fusion gene in individual cases of ARMS, formed by t(2;2)(q35;p23) translocation [5].Hajime et al. found that tumors with ERMS have a novel PAX3 rearrangement, formed by t(2;8)(q35;q13) translocation [6]. And it has been suggested that the new PAX3 rearrangement is the cytogenetic basis of congenital ERMS [7]. Other researchers have found der( 16) t( 1; 16) translocations in ERMS [ 8].
Almost all RMS (94%) show at least one 15mb long heterozygous deletion (LOH) region. The most common LOH region is located on chromosome 11. the short arm of chromosome 11 (11p15. 5) LOH has been considered as a feature of ERMS [ 5]. suggesting the presence of inactivation of oncogenes. Molecular biology studies suggested a 100-fold increase in IGF2 (insulin-like growth factor-2) in patients with both subtypes of RMS, which was significantly higher than in control normal skeletal muscle, while there was no correlation between IGF2 mRNA expression and LOH in the 11p15 region, but samples with RMS with LOH showed a tendency for high IGF2 mRNA expression. Moreover, MyoD1 mRNA had similar levels of high expression in both subtypes and in common skeletal muscle, and its high expression correlated with LOH. myogenin mRNA expression was not statistically significant in the RMS subtype [9]. It has also been shown that Snail transcription factors and Snail-related genes are differentially expressed in follicular and embryonic RMS. snail is highly expressed in ARMS patient samples and in vesicular cell lines, and E-Cadherin protein expression is downregulated and MMP2, MMP9 protein expression is upregulated in ARMS patient samples. Therefore, it is inferred that Snail gene plays an important role in tumor metastasis as a regulator in many tumors and RMS by down-regulating E-Cadherin protein expression and up-regulating metalloprotein expression. The findings also explain the more aggressive nature of ARMS and even infer that further studies will demonstrate that Snail may serve as a potential target for ARMS therapy [10].
P53 pathway inactivation is the most common gene pathway alteration in human tumors and can arise from the p53 mutation itself or from other alterations such as increased MDM2 expression (regulating p53 downregulation) or upstream mediators (e.g. CDKN2A). rms cell lines have a high frequency of p53 mutations. gene sequencing studies of rms cell lines have shown that exons 7 and 8 of the p53 gene are mutated The frequency of mutations in exons 7 and 8 of the P53 gene was 8% and 14%; changes in intron 7 were more pronounced, including lamellar deletions, insertions, shifts and point mutations, and these alterations had no similarity to the wild-type p53 sequence. Studies have shown that inactivation of oncogenes may play an important role in the malignancy of embryonic cells in vitro [11]. Deletion of the P53 gene in mature myoblasts predisposes them to ERMS [12]. p53 gene mutations can occur at early or late tumor stages, and expression of mutant p53 genes is usually accompanied by a more aggressive tumor biology. Expression of mutant p53 suggests a poor prognosis for RMS. MYCN is one of the direct target genes upregulated by PAX-FKHR. MYC genes are an early group of oncogenes identified, including c-myc, n-myc and -lmyc. The myc gene family and its products promote cell proliferation, immortalization, dedifferentiation and transformation, and are important in many tumorigenic processes. In patients with ARMS, patients overexpressing MYCN genes have a poorer prognosis, and MYCN abnormalities are characteristic of RMS development and can distinguish patients with poorer prognosis as a potential target for new therapies [ 13].
Chromosomal translocations, LOH and oncogene gene inactivation, and oncogene amplification lead to changes in the corresponding proteins through alterations in the corresponding signaling pathways, leading to the development and progression of RMS. Currently, the related molecular pathways include PAX3/PAX7-FKHR, IGF2 signaling system and IL-4 pathway.
2.Pathology of RMS
2.1 Pathological features
Pathologically, RMS is a blue small round cell tumor, and its histological classification includes embryonal rhabdomyosarcoma [(embryonal rhabdomyosarcoma, ERMS) accounting for 80% of all RMS, including the spindle cell grape-like variant] and adenosarcoma [(alveolar rhabdomyosarcom a, ERMS has small cytosomes, little cytoplasm, deep-stained round nuclei, and indistinct nucleoli. The tumor cells were diffusely distributed, and some areas were unevenly spaced. There were a few cells with slightly more cytoplasm, red staining and deviated nuclei. The ARMS microscopy reveals small round or oval cells separated by fibrous connective tissue into nests, resulting in a typical glandular and pseudoglandular pattern. The tumor cells in PRMS are abundant, extremely diverse in size and morphology, i.e., tumor cells of different differentiation stages can be seen. Most of the tumor cells are mixed, with no specific arrangement[14] .
Masson’s trichrome staining can show microfibrils and transverse lines well. Most scholars now believe that the longitudinal myofilaments surrounding the cytoplasm and distributed in concentric circles detected in Masson trichrome staining are valuable for the diagnosis of rhabdomyosarcoma. Silverophilic staining reveals sparse reticulate tissue fibers and intercellular collagen. Immunohistochemical staining: Four antibodies against vimentin, desmin, smooth muscle actin (SMA), and myoglobin are used. Vimentin is a specific marker of mesenchymal normal cells and tumors, mainly used to distinguish cancer from sarcoma, with high positive rate and poor specificity. Desmin is a cytoskeletal protein found in smooth muscle and skeletal muscle, but is valuable in the diagnosis of rhabdomyosarcoma. myoglobin is widely found in cardiac and skeletal muscle and is highly specific for rhabdomyosarcoma and its tumors, but is less sensitive. Chen Wei et al. reported that the combination of four antibodies was the most effective, and the diagnosis was confirmed in most cases in combination with light microscopic features [15]. Wang Zheng et al. reported that Caveolin-3 expression in RMS has high sensitivity and specificity and can be a useful novel marker for clinical differential diagnosis of RMS and other soft tissue tumors [16].Caveolin 1 (niche protein) can be used as a marker for hypodifferentiated rhabdomyosarcoma [17]. Recent studies have shown the expression of Glypican-3 (phosphatidylinositol [protein] glycan-3) in RMS, but not in other sarcomas, so it could be used as a biomarker for this disease in the future [18]. In addition muscle-specific miRNAs exist and miR206 expression correlates with the degree of muscle differentiation. Standardized miR-206 expression levels can be used to distinguish rhabdomyosarcoma from non-rhabdomyosarcoma with a sensitivity of 1.0 and specificity of 0.913, therefore the application of muscle-specific miRNAs, especially miR-206, can be used as a cut-off marker for biological markers of rhabdomyosarcoma [19].
2.2 Pathological staging
The choice of tumor treatment plan and prognosis depends heavily on the determination of the degree of local tumor infiltration and metastasis. At present, there are various staging methods in common use internationally, which are not exactly the same. Currently, the Intergroup Rhabdomyosarcoma Study (IRS ( IV) classification is mostly used, which is useful for clinical prediction. The IRS clinical staging of rhabdomyosarcoma is as follows
Stage I: tumor confined, completely resected, and regional lymph nodes not involved.
Ⅰa tumor confined to the primary muscle or primary organ
Ⅰb tumor invades into adjacent tissues other than the primary muscle or organ, such as through the fascial layer
Stage II: The tumor is confined, completely resected by the naked eye with or without microscopic residue. (complete resection of the tumor as seen by the naked eye, with local invasion or regional lymph node metastasis).
IIa complete resection of the tumor with microscopic residue and no metastasis of regional lymph nodes
Ⅱb complete resection of tumor with no microscopic residue, but regional lymph node metastasis
IIc complete resection of tumor with microscopic residue, but regional lymph node metastasis
Stage III: Incomplete resection or biopsy only, with microscopic residue in the primary focus or regional lymph nodes. (Stage II tumor is not completely resected or only biopsy sampling is performed, and there is residual tumor in the naked eye).
IIIa Biopsy sampling only
IIIb Most of the tumor seen by the naked eye is removed, but there is obvious residual tumor to the naked eye
Stage IV: distant metastasis at the time of diagnosis (the tumor already has distant metastasis, such as lung, liver, bone marrow, brain, distant muscle or lymph node metastasis at the time of diagnosis).
3.Clinical manifestation of RMS
The annual incidence of rhabdomyosarcoma is 0.4414 per 100,000. The male:female incidence rate is 3:2. 67% of the cases are embryonal rhabdomyosarcoma, 32% are glandular rhabdomyosarcoma, and 1% are pleomorphic rhabdomyosarcoma. The glandular follicular and pleomorphic RMS were more often seen in adults, whereas the embryonal type was more often seen in children (p = 0.0001) [20]. The different tissue types are closely related to the age of onset and site. The embryonic type occurs overwhelmingly in children aged 3 to 12 years and is the most common type of RMS. It predominantly occurs in the head and neck and genital tract. The gravid type occurs in cavernous organs, such as the genitourinary and nasopharyngeal tracts and the biliary tract, and is common in infants and young children. The glandular vesicle type is more common in young people, with the extremities, head and neck, and perineum/perineum as the most common sites. The polymorphic form is more likely to occur in adults, with a predilection for the extremities and, to a lesser extent, the trunk [21]. The disease is highly malignant and is clearly aggressive, causing widespread metastases along blood vessels and lymphatic vessels at an early stage. The disease has a short course and is most often seen within six months. The main symptoms are painful or painless masses, and pain can occur when the tumor compresses nerves. The skin surface is red and swollen, and the skin temperature is high. Tumor size varies, hard texture, and most of the masses are fixed at the time of consultation. When the tumor grows fast, there may be skin rupture and bleeding. Head and neck masses may have protruding eyes, bloody discharge, nasal bleeding, swallowing and breathing disorders. RMS mostly metastasizes to retroperitoneal lymph nodes and lymph nodes in the region to which it belongs, and in advanced stage, it is often accompanied by hematogenous metastasis. The combination of pathology, imaging, bone scan, bone marrow cytology and other examinations can assist in diagnostic staging and clinical staging.
4.Diagnosis of RMS
The clinical manifestations of transverse myosarcoma are non-specific, so early diagnosis is difficult and easily misdiagnosed as benign lesions, and the definite diagnosis depends on surgical pathology. Histopathologically, it usually appears as poorly differentiated small round cells, which do not have the morphological characteristics of rhabdomyoblasts. Therefore, pathologically, RMS is difficult to differentiate from small round cell tumors commonly seen in children, such as malignant lymphoma, neuroblastoma, Ewing’s tumor, and primitive neuroectodermal tumors. The immunohistochemical staining with antibodies against myogenic regulatory factors and the observation of primitive Z-band with mixed thick and thin myofilaments and myofilaments under the combined electron microscope are of great significance for the definite pathological diagnosis, and CT, magnetic resonance, bone scan and other techniques provide the basis for judging the stage, nature and infiltration range of the tumor and guiding the surgical treatment.
5.Treatment and prognosis of RMS
The treatment of RMS includes systemic treatment and local treatment. Most patients with ERMS have already developed subclinical metastases at the time of consultation. Even if surgery or radiotherapy effectively controls the local tumor, some patients still have recurrence within a few months. Incomplete treatment of the primary tumor also seriously affects the prognosis. Recently, early combination chemotherapy before surgical resection is advocated to eliminate subclinical metastases and reduce the size of the primary tumor to facilitate surgical resection. Combination chemotherapy reduces the activity of tumor cells and decreases the dissemination of tumor cells during surgery. According to the tumor stage, the strict formulation of a comprehensive plan of surgery and radiotherapy is the key to improve the prognosis. For RMS, the first-line chemotherapeutic agents are: cyclophosphamide, vincristine, cisplatin, amoxicillin and radiolucent D. Topotecan (topotecan), irinotecan (irlnotecan) and paclitaxel (paclitaxel) can be used as second-line agents for RMS chemotherapy. Some studies suggest that radiotherapy can be dispensed with in children with stage I embryonic type, while the remaining stages I to III can be treated with radiotherapy to improve the long-term outcome by 25-30%; for stage IV patients overdose fractionated irradiation can be used with less impact on the long-term prognosis.Million L et al. showed that three-quarters of children died when local tumors were not controlled, emphasizing the importance of applying radiotherapy in children with stage II rhabdomyosarcoma [22 ]. In children too young (<3 years) to receive radiation therapy, overdose combination chemotherapy supplemented with hematopoietic stem cell transplantation is used to reduce chemotherapy response and delay the start of radiation therapy. Zhang Yi et al. reported that the use of autologous peripheral stem cell transplantation in children with stage III ERMS resulted in better outcomes and high long-term survival rates [ 23].
The molecular biology of ERMS has been studied, making immunotherapy possible. Proteins encoded by specific genes within tumor cells are presented on the cell surface as peptides, and this specific encoded protein can be a target for cytotoxic T cell action. For example, peptides of the mutant p53 protein product have been found to be specifically recognized by cytotoxic T cells, and this approach has been applied in the immunotherapy of children with alveolar rhabdomyosarcoma [24]. mi-63 is an effective therapeutic factor for ERMS expressing wild-type P53 protein and has been shown to have a coordinated effect with doxorubicin [25]. Genetic analysis suggests that the expression of epidermal growth factor receptor (VEGFR) is significantly higher in ERMS than in ARMS, and in vitro studies have demonstrated that its specific antibody Cetuximab binds ERMS in tumor cell lines and can be detected by flow cytometry. Thus, such antibodies would provide less cytotoxic immunotherapy for ERMS and could be used in the treatment of drug-resistant ERMS [26].Seitz G et al. found that multiple resistance mechanisms in rhabdomyosarcoma are mediated through genes and proteins of the GST family. Reversal of these effects may be obtained through partial GST suppressors. Thus, the GST family offers a promising target for drug-resistant treatment strategies in pediatric RMS [27].
RMS is a malignant solid tumor with an overall poor prognosis. Studies have shown that the most common pediatric ERMS have a better prognosis than ARMS and PRMS. Embryonic histological typing, favorable tumor site, age less than 10 years and absence of distant metastases and surgical resection significantly improve survival [20]. molecular biology studies of RMS suggest a correlation between its molecular biology and clinical prognosis, therefore adenoviral vector-mediated gene therapy and immunotherapy would offer greater hope for children with rhabdomyosarcoma and improve their prognosis.