Ewing’s Sarcoma/ Peripheral Primitive Neuroectodermal Tumor: A Case Report and Review of Literature
Introduction
Ewing’s sarcoma Family of Tumors (EFTs)
EFTs represent the second most common primary bone malignancy, after osteosarcoma, affecting children, adolescents, and young adults. [2]
EFTs is a group of cancers that includes (a) the Ewing Sarcoma (EWS), (b) the extraosseous Ewing (EOE) tumors, (c) the primitive neuroectodermal tumors (PNET) and (d) Askin tumors. [1] EWS accounts for approximately 70% of the tumors in the family. [4]
These tumors are encompassed into one family because most of them are associated with the same chromosomal abnormality: a non-random, balanced reciprocal translocation between a portion of the EWS gene of the 22nd chromosome and a portion of the FLI-1 gene of the 11th chromosome [t(11;22)(q24;q12)] [3][4].
Individuals with a neoplasm from the EFTs can experience pain, swelling, and tenderness near the affected bone or soft tissue area. Pain is often intermittent at first, but it becomes progressively more consistent. Fever, fatigue, weight loss, low levels of circulating RBCs, and increased levels of circulating WBCs are symptoms that these individuals may experience. [4]
The diaphyseal region of the long bones of the arms and legs, particularly the femur, is most affected by EFTs. The trunk and chest are the most common sites for soft tissue tumors. [2][4]
Histologically, approximately 70% of EFTs cases can be characterized by densely packed small-round-blue cells with hyperchromatic nuclei, limited but clear cytoplasm (due to glycogen presence), concealed nucleoli and no ECM production. [6]
Some secondary features include lobule-like presentation, excessive cell necrosis, epithelial patterns indicating the presence of a blood vessel nearby, biphasic population of light-and-dark tumor cells and filigree patterns (intercellular stromas). [6]
Ewing’s Sarcoma (EWS)/ peripheral Primitive Neuroectodermal Tumor (pPNET)
PNETs and especially pPNETs is a group of highly aggressive malignancies, characterized by the distinct small cells of the EFTs with neuroectodermal origin that affect bone and soft tissue exclusively. They exhibit great diversity in their clinical manifestations and many similarities with other members of the EFTs. As a result, classifying these groups has been difficult and controversial. [7]
However, the overlapping morphologic features of EWS and pPNET, such as their ultrastructure, immunophenotype, and a common set of molecular-genetic abnormalities, have recently led to the conclusion that they practically follow the same neoplastic process and therefore, essentially considered the same. [5]
The preferred terminology of “EWS/pPNET” was proposed by the World Health Organization (WHO) [8]
EWS/pPNET accounts for approximately 70% of the tumors in the EFTs. [4] In the prevalence of soft tissue and bone tumors, there are some unexplained ethnic, racial, and sexual disparities, including slight male predominance (1.0:1.6). [2][3]
Histologically, except from the characteristic features of the EFTs, EWS/pPNET can be distinguished by the presence of Homer-Wright rosettes (differentiated cells grouped around a central region containing neuropil) . [6]
The presence of Homer-Wright rosettes and/or immunopositivity of two neural markers has been used in the past to differentiated EWS/pPNET from other EFTs. [6]
It must be noted that distinguishing EWS/pPNET from lymphoma has always been a challenge. [9] This highlights the importance of histopathology, which is the fact that it enables experts, on a more general scale, to explore histological abnormalities that can clarify the real cause of the patient’s illness. Pathologists can reach a conclusion for their diagnosis, just by examining tissue from the relevant areas.
Materials and Methods
Patient’s Clinical History
Patient is a 15-year-old female with a 2-month history of increasing lower back pain and bilateral leg pain. Patient presented herself to the ICU due to urinary retention and inability to walk. After a series of physical tests, the following conclusion can be made: presence of a neurological issue (positive Babinski reflex) causing sensory ataxia (positive Romberg reflex) with high probability of it incorporating the corticospinal tract (positive Hoffman reflex) but low probability of it caused by injuries to the specific spinal reflex arcs (S2–S4) which innervate the pudendal nerve (positive bulbocavernosus reflex). Radiographic techniques indicated the presence of an intraspinal, extradural, 6.5X0.8X2.3cm mass extending from T3 to T7. The mass demonstrated its involvement in the bilateral transverse process, the spinous process, the spinal canal, and the neural foramina. Due to presented spinal compression, a decompression surgery was performed. [9]
Methodology
T1 weighted MRI scan with Gadolinium: Contrast agents are used in MRI to make internal body structures more visible. In this case gadolinium-based contrast agent was used. Note that these agents shorten the T1 relaxation time of protons inside tissues and thus, T1 time is restored back to normal. [3]
Figure 1: Radiography: MRI This figure represents (a) Sagittal T1 weighted MRI scan post Gadolinium (Gd) using fat saturation (Figure 1A). (b) Axial T1 weighted MRI scan post Gd at T5 (Figure 1B). (c) Axial T1 weighted MRI scan after Gd (Figure 1C, scan was taken at T5).
Intraoperative Tissue Preparation: Frozen Section Preparation: The pathologist was asked by the surgeon to immediately evaluate the tumor tissue obtained during surgery, because instant pathologic diagnosis could determine whether and how the surgery was going to proceed. The evaluation method employed was frozen tissue preparation. The surgeon removed a portion of the tissue mass during the surgery. After that, the biopsy was sent to the pathologist. The tissue was frozen in a cryostat machine, then cut with a microtome and stained with various dyes, including H&E and DiffQuick (Figure 2A), so that it can be examined under a microscope. The procedure took only a few minutes and that’s why it was preferred. [9][6]
Postoperative Tissue Preparation: Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Preparation: Cytologic preparation was also done via the FFPE tissue preparation. To preserve the proteins and vital structures within the tissue, the sample was first fixed in formalin (formaldehyde). It was then embedded in paraffin wax blocks which made cutting slices of the required size much easier. Finally, the sample was H&E stained for it to be examined (Figure 2B&2C) [3][9].
Some of the FFPE tissue samples were extensively tested by several immunoreactive agents, including CD99 (Figure 2D), S-100 protein, HMB45 and leukocyte common antigen.
Postoperative Tissue Preparation: Resin-embedded Section Preparation: Resins are the most common embedding medium for electron microscopy studies. The preparation for embedding with resins is very similar to that performed for FFPE, but with some modifications. After being fixated and embedded in paraffin, liquid resin was added to make the tissue sample solid. Polymerization of resins proceeded at 60 ºC which made the sample ready for microscopic examination. (Figure 3A&3B) [6]
Fluorescent in situ hybridization (FISH): FISH is a molecular cytogenetic technique that employs fluorescent that detects and locates the presence of specific DNA sequences on chromosomes. Fluorescence microscopy was used (Figure 2D, insert) to reveal the fluorescent probe bound to the examined for abnormality, chromosomes (see 1.1). [5]
Results
The decompression surgery yielded a 3.5X3.0X0.8cm aggregate of soft tissue fragments. Frozen section preparation revealed a population of small blue cells with round and centrally located hyperchromatic nuclei. Their cell membranes were distinct; their cytoplasm was granular (Figure 2A) and contained small cytoplasmic vacuoles (Figure 2A, arrows). These vacuoles were best visualized with DiffQuick stain (Figure 2A, insert) as they could not be well appreciated on frozen sections. [9]
FFPE sections showed tumor invasion into the bone and the fibroadipose tissue (Figure 2B). The tumor cells were small to medium sized and had mild variation in nuclear size with the smaller ones hyperchromatic to the larger ones. Mitotic figures were relatively common. The cytoplasm had small cytoplasmic vacuoles (Figure 2C) that contained substantial amount of PAS positive (Figure 2C, insert) enzyme-sensitive material.
The tumor cells demonstrated strongly positive membranous immunoreactivity for vimentin, CD99 (Figure 2D) but no immunoreactivity for S-100 protein, HMB45 and LCA.
FISH demonstrated fusion signals consistent with reciprocal translocation of t(11;22)(q24;q12) (Figure 2D, insert). The fusion gene, EWS/FLI-1, is well demonstrated in this case; FISH shows fusion (yellow or red-green signals) of the FLI-1 gene (green signals) with EWS gene (red signals) (Figure 2D, insert). [9]
The cytoplasmic vacuoles were well demonstrated in resin embedded section (Figure 3, insert). Ultrastructurally, the tumor cells had scattered, unnoticeable cytoplasmic organelles and prominent non-membrane bound cytoplasmic vacuoles that were lacking organelles (Figure 3A). Some of these vacuoles contained residual glycogen particles (Figure 3B). [9]
Figure 2: Histology of the EWS/pPNET tissue (light microscopy) This figure represents histological tissue samples taken intraoperatively from the tumor. (a) Frozen section, H&E (Figure 2A) (b) Frozen section, H&E, DiffQuick (Figure 2A, insert) (c) PPFE, H&E (Figure 2B) (d) PPFE, H&E (Figure 2C) (e) PPFE, H&E, PAS (Figure 2C insert) (f) PPFE, CD99 (Figure 2D) (g) FISH (Figure 2D, insert).
Figure 3: Histology of the EWS/pPNET tissue (electron microscopy) This figure represents histological tissue samples taken intraoperatively from the tumor. Both images were prepared by resin-embedded section preparation.
Discussion
Diagnosis
First and foremost, the small bluish cells with hyperchromatic nuclei (Figure 2A& 2A insert) indicate presence of neoplastic cells which belongs to the EFTs. [9]
The clarity of the cell membranes has not been consistently correlated with differential diagnosis in relation to the EFTs [6]
The fact that small granules can be observed in the cytoplasm of the cells (Figure 2A insert) along with the PAS positive staining (Figure 2C insert) which translates into the presence of glycogen, indicates the presence of small granules which secrete glycogen, characteristic of the EFTs, once again. [8]
Similarly, under the electron microscope, tumor cells showe scattered prominent vacuoles, some of those contained glycogen (Figure 3), contributing to the same indication.
Continuing, the tumor invasion into the nearby bone and fibroadipose tissue was observed in a PPFE section (Figure 2B), which is an indication that the cells belong specifically to the EWS/pPNET subgroup. [7]
The presence of small cells with mild size variation, with the smaller ones being more hyperchromatic, creating this biphasic dark-and-light appearance (Figure 2C, cell on the left being darker than those on the right), is another characteristic feature of cell in the EFTs [6].
Also, the absence of lymphoglandular bodies in any paraffin-embedded finding (Figure 2), is a sign that this is not a case of lymphoma.
Additionally, FISH demonstrated fusion signals consistent with the characteristic translocation of the ETFs, t(11;22) (q24;q12) (Figure 2D, inset).
Finally, in certain paraffin-embedded sections (Figures 2C and 2C insert), we can observe the presence of neuropil, which forms the Homer-Wright rosettes similar to those seen in neuroblastoma and perivascular pseudorosettes. [3]
As stated in the introduction (see 1.2), the only established way to differentiate EWS/pPNET from other EFTs is either via the presence of Homer-Wright rosettes and/or immunopositivity of two neural markers. [6]
Moving on to the immunohistology, the tumor cells demonstrated strong positive membranous immunoreactivity for vimentin (CD99) (Figure 2D) but no immunoreactivity for S-100 protein, Human Melanoma Black 45 (HMB45) antibodies and leukocyte common antigen (LCA).
EFTs are typically immunoreactive to the following agents: CD99, caveolin 1 (CAV1), and friend leukemia integration 1 (FLI1). CD99 is positive in 99–100% of ETFs cases where molecular confirmation is available but it can be positive in other small-round-blue cells tumors. Immuno-expression of those agents is common but not very specific and should be compared to a panel of negative markers, such as LCA. Antibodies such as HMB-45 and S-100 protein have been used in combination to enhance the detection of metastatic melanoma. [6]
Thus, we can once again confirm that is a case of EFTs (positive CD99) and not a case of either melanoma (negative HMB-45 and S-100) or lymphoma (negative LCA).
Exclusion of other tumors in the differential diagnosis and correlation with clinical, morphological, and immunohistochemical features are required for a correct diagnosis. That was the only way to conclude that this is a rare case of EWS/pPNET.
Etiology
No specific environmental exposures have been identified as risk factors for development of these tumors. They are thought to arise sporadically. [4]
The only genetic correlation is the fact that most of subgroups inside the EFTs are associated with the same chromosomal abnormality, t(11;22)(q24;q12) [3][4].
Management
Chemotherapy and local control, either by surgery, radiation therapy, or a combination of both, are used to treat EWS/pPNET. [4]
Before treating the primary tumor, a multi-agent chemotherapy protocol is applied for several cycles. If the cancer is not responding, surgical resection is recommended. Radiation alone can be used if the tumor cannot be resected. [4][3]
As long as complete tumor resection is anatomically possible, limb salvage surgical resection can improve the best functional. In some cases, however, amputation can provide the best functional outcome. [4]
Endoprostheses are frequently used to repair bone defects caused by the surgical resections. Expandable prostheses have been specifically developed for immature children's reconstruction and allow for limb lengthening, allowing for limb salvage surgery in the younger skeletally immature patient. [4]
References
National Cancer Institute. (2022). Ewing Sarcoma and Undifferentiated Small Round Cell Sarcomas of Bone and Soft Tissue Treatment (PDQ®)–Health Professional. URL: https://www.cancer.gov/types/bone/hp/ewing-treatment-pdq
Henock T. Wolde-Semait, Scott Rosenfeld (2017) Ewing's Sarcoma/ Peripheral Neuroectodermal Tumor, Study Guide. Pediatric Orthopedic Society of North America URL: https://posna.org/Physician-Education/Study-Guide/Ewing-s-Sarcoma-Peripheral-Neuroectodermal-Tumor
Maria Tsokos, Rita D. Alaggio, Louis P. Dehner, and Paul S. Dickman. (2020). Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumor and Related Tumors. HHS Author Manuscripts. URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993191/
Patrick J. (unavailable). Ewing’s Sarcoma. Grohar National Organization for Rare Disease. URL: https://rarediseases.org/rare-diseases/ewing-sarcoma/
L P Dehner. (1993). Primitive neuroectodermal tumor and Ewing's sarcoma. Am J Surg Pathol. URL: https://pubmed.ncbi.nlm.nih.gov/8383465/
Hue-Tsi Wu (2011) Dhirendra Govender Ewing sarcoma family of tumours: unusual histological variants and immunophenotypic characteristics. Diagnostic Histopathology URL: https://www.sciencedirect.com/science/article/pii/S1756231712000989#!
Carlo P Honrado (2013) Primitive Neuroectodermal Tumors. Otolaryngology and Facial Plastic Surgery. Medscape URL: https://emedicine.medscape.com/article/855644-overview#:~:text=Peripheral primitive neuroectodermal tumors (pPNETs) are a group of aggressive,in the head and neck
ISBN of Soft Tissue and Bone Tumours, WHO Classification of Tumours, 5th Edition, Volume 3
Any information noted with the citation [9] are indicatory material taken from the “207-Histology” subject.
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