This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS048353 and R01 NS109655 to S.L.C.; R01 NS109655-03S1 to D.P.J.), the National Cancer Institute (R01 CA122804 to S.L.C.) and the Department of Defense (X81XWH-09-1-0086 and W81XWH-12-1-0164 to S.L.C.).

Pathology Assessment of PN-MPNST Progression in the P0-GGFβ3 Mouse Model

<ol>
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The authors have no conflicts of interest to disclose.

The histological and biochemical methods presented here provide a framework for diagnosing and characterizing GEM models of neurofibroma and MPNST pathogenesis. Over the years, we have found these methodologies to be quite useful for assessing the pathology of peripheral nerve sheath tumors arising in GEM models21,25,26. However, while the protocols outlined here are useful for determining how accurately tumors in the GEM models...

Over the last three decades, numerous laboratories have attempted to create mouse models of human cancers by introducing human cancer-associated mutations into the mouse genome or by overexpressing a gene product that is overexpressed in human cancers. The resulting genetically engineered mouse (GEM) models can be used for a variety of purposes such as establishing that the newly introduced genomic modification initiates tumorigenesis, identifying other subsequently occurring genetic or epigenetic changes that contribute to tumor progression, and defining the key signaling pathways that drive tumor initiation and progression. Unlike orthotopic xenograft models, which rely on the use of immunodeficient mice, GEM cancer models have a fully functional immune system and so more accurately model responses to candidate therapeutic agents. However, when using GEM cancer models for purposes such as these, it is essential that investigators confirm that observations made with GEM neoplasms are relevant to their human counterparts. This validation should include a thorough assessment of the pathology of the GEM neoplasms and a determination as to whether the pathologic features of the GEM neoplasms recapitulate the pathology of the corresponding human tumor type.
The tumor susceptibility syndrome neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the human nervous system, occurring in approximately 1 in every 3,000-3,500 live births1,2,3. Individuals afflicted with NF1 develop multiple benign peripheral nerve sheath tumors known as neurofibromas in their skin (dermal neurofibromas) and in large nerves and nerve plexuses (plexiform neurofibromas). While both dermal and plexiform neurofibromas worsen the patient&#39;s quality of life by producing physical, behavioral, and/or social impairment, plexiform neurofibromas (PNs) are particularly dangerous4,5.&#160;This is because PNs frequently transform into malignant peripheral nerve sheath tumors (MPNSTs), which are aggressive spindle cell neoplasms with an exceptionally low survival rate1,2. In large part, this low survival rate is because the radio- and chemotherapeutic regimens that are currently used to treat MPNSTs are ineffective. However, developing new, more effective therapies has been challenging. This is because, despite how commonly MPNSTs occur in NF1 patients, they are still rare neoplasms. As a result, it is very difficult to obtain large numbers of human tumors for study; it is also challenging to recruit enough patients with MPNSTs for clinical trials. To overcome these limitations, several GEM models have been generated with the goal of gaining further insights into the abnormalities driving neurofibroma pathogenesis and PN-MPNST progression and to facilitate preclinical trials with candidate therapeutic agents.
NF1 patients have inactivating mutations in one copy of the NF1 gene. Neurofibroma pathogenesis is triggered when an inactivating mutation in the remaining functional NF1 gene occurs in a cell in the Schwann cell lineage. Surprisingly, however, when mice were generated with germline inactivating Nf1 mutations, they did not develop neurofibromas6,7. The subsequent demonstration that mice with Nf1-null Schwann cells and Nf1 haploinsufficiency in all other cell types (Krox20-Cre;Nf1flox/- mice) developed plexiform neurofibromas suggested that reduced Nf1 gene dosage in additional cell types was required for neurofibroma pathogenesis8. Even then, the plexiform neurofibromas in Krox20-Cre;Nf1flox/- mice did not progress to become MPNSTs and so only partially mimicked the biology of their human counterparts. MPNST pathogenesis did occur when Nf1 mutations were partnered with mutations in additional tumor suppressor genes such as Trp539 or Cdkn2a10, but MPNSTs in these GEM models developed de novo or from atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs)11,12, rather than from preexisting benign plexiform neurofibromas (see13,14 for excellent reviews of these models as well as other models introducing additional MPNST-associated loss of function mutations in genes such as Suz12 and Pten15).
These mouse models have been invaluable for establishing the role that genes such as NF1, TP53, and CDKN2A play in the pathogenesis of NF1-associated peripheral nervous system neoplasms and for preclinical trials testing candidate therapeutic agents. However, we still have an incomplete understanding of the process by which plexiform neurofibromas progress to become atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs16) and then MPNSTs. Some progress has recently been made in understanding this process with the recent report that mice with deletions in Nf1 and Arf develop ANNUBPs that progress to become MPNSTs11. However, Nf1 mutation-based mouse models that fully recapitulate the process of plexiform neurofibroma-MPNST progression seen in humans do not yet exist. In addition, it is not clear whether there are multiple distinct pathways that lead to the development of MPNSTs. Given this, it is possible that the GEMs described above only model a subset of several different pathways that lead to neurofibroma-MPNST progression and MPNST pathogenesis. This point is emphasized by the fact that MPNSTs also occur sporadically and that some sporadic MPNSTs apparently do not have NF1 mutations17,18.
Although this latter point has been challenged by Magollon-Lorenz et al.&#39;s recent suggestion that at least some sporadic MPNSTs lacking NF1 mutations are melanomas or a different type of sarcoma19, we have recently reported a sporadic MPNST and a cell line derived from this tumor (2XSB cells) that was NF1 wild-type20. During the characterization of the parent tumor and the 2XSB cell line, we systematically ruled out alternative diagnostic possibilities, including melanoma and the multiple other sarcoma types that are routinely considered in the differential diagnosis of a sporadic malignant peripheral nerve sheath tumor20. In addition, we note that Magollon-Lorenz et al. acknowledged that their findings in the three sporadic MPNST cell lines that they studied could not be generalized to indicate that all tumors identified as sporadic MPNSTs are not MPNSTs.
To construct a GEM model in which neurofibroma and MPNST pathogenesis were not necessarily dependent upon specific tumor suppressor gene mutations, we generated transgenic mice in which overexpression of the potent Schwann cell mitogen neuregulin-1 (NRG1) was driven by the Schwann cell-specific myelin protein zero (P0) promoter (P0-GGF&#946;3 mice)21. We have previously shown that human neurofibromas, MPNSTs, and MPNST cell lines express several NRG1 isoforms together with the erbB receptor tyrosine kinases (erbB2, erbB3, and erbB4) that mediate NRG1 signaling and that these erbB receptors are constitutively activated22. We have also demonstrated that pharmacologic inhibitors of the erbB kinases potently inhibit MPNST proliferation22, survival23, and migration24. In keeping with our observations in humans, P0-GGF&#946;3 mice develop plexiform neurofibromas25 that progress to become MPNSTs at a high frequency21,25. We have shown that P0-GGF&#946;3 MPNSTs, like their human counterparts, commonly develop mutations of Trp53 and Cdkn2a, as well as numerous other genomic abnormalities that potentially contribute to tumorigenesis25. MPNSTs arising in P0-GGF&#946;3 mice do not have inactivating Nf1 mutations. However, using genetic complementation, we showed that NRG1 promotes tumorigenesis in P0-GGF&#946;3 mice predominantly through the same signaling cascades that are altered by Nf1 loss26; this conclusion is based on our finding that substituting NRG1 overexpression for Nf1 loss in the presence of Trp53 haploinsufficiency (P0-GGF&#946;3;Trp53+/- mice) produces animals in which MPNSTs develop de novo, as is seen in cis-Nf1+/-;Trp53+/- mice27.
To obtain this and other information demonstrating that P0-GGF&#946;3 mice accurately model the processes of neurofibroma pathogenesis and neurofibroma-MPNST progression seen in humans with NF1, we have developed specialized methodologies for processing tissues from these animals, accurately diagnosing their tumors, grading the MPNSTs arising in these mice, establishing and characterizing early-passage P0-GGF&#946;3 and P0-GGF&#946;3;Trp53+/- MPNST cultures and critically comparing the pathology of P0-GGF&#946;3 PNs and MPNSTs and P0-GGF&#946;3;Trp53+/- MPNSTs to that of their human counterparts. Many of these methodologies are generalizable to other GEM models of nervous system neoplasia. Additionally, several of these methodologies are more broadly applicable to GEM models in which neoplasms arise in other organ sites. Consequently, here we present a detailed description of these methodologies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Tissue Culture Plates</td>
			<td>Corning Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;- Diaminobensidine (DAB)</td>
			<td>Vector Laboratories</td>
			<td>SK-400</td>
			<td></td>
		</tr>
		<tr>
			<td>6- well plates</td>
			<td>Corning Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher Scientific</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Secondary (Goat Anti-Mouse)</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Secondary (Goat Anti-Mouse)</td>
			<td>Invitrogen</td>
			<td>A21043 or A11004</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Secondary (Goat Anti-Rabbit)</td>
			<td>Invitrogen</td>
			<td>A11036</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride (NH4Cl)</td>
			<td>Fisher Scientific</td>
			<td>A661-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Caldesmon</td>
			<td>ABCAM&#160;</td>
			<td>E89, ab32330</td>
			<td></td>
		</tr>
		<tr>
			<td>CD117</td>
			<td>Cell Marque</td>
			<td>117R-18-ASR</td>
			<td></td>
		</tr>
		<tr>
			<td>CD163</td>
			<td>Leica</td>
			<td>NCL-L-CD163</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>ABCAM&#160;</td>
			<td>ab29364</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>ABCAM&#160;</td>
			<td>ab81289</td>
			<td></td>
		</tr>
		<tr>
			<td>CD86</td>
			<td>ABCAM&#160;</td>
			<td>ab53004</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Stripper</td>
			<td>Corning</td>
			<td>25-056-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-545-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrisolve Hybrid (d-limonene-based solvent)</td>
			<td>Decon Laboratories</td>
			<td>5989-27-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Critic Acid</td>
			<td>Fisher Scientific</td>
			<td>A104-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin</td>
			<td>ABCAM&#160;</td>
			<td>C-11, ab7753</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmin</td>
			<td>Agilent Dako&#160;</td>
			<td>clone D33 (M0760)</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobensizdine (DAB) Solution</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Thermo Scientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (200 Proof)</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Omega Scientific</td>
			<td>FB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Forksolin</td>
			<td>Sigma-Aldrich</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G6279</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Fisherbrand</td>
			<td>245-677</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Brightline-Hauser Scientific</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>327-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Iba1</td>
			<td>Wako Chemicals</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPRESS HRP (Peroxidase) Polymer Kit ,Mouse on Mouse</td>
			<td>Vector Laboratories</td>
			<td>MP-2400</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPRESS HRP (Peroxidase) Polymer Kit, Horse Anti-Rabbit</td>
			<td>Vector Laboratories</td>
			<td>MP-7401</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>Heracell 240i CO2 incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A415</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki-67</td>
			<td>Cell Signaling&#160;</td>
			<td>12202</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MART1</td>
			<td>ABCAM&#160;</td>
			<td>M2-9E3, ab187369</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore&#160;</td>
			<td>Human: MAD5236 (10C2), Human:MAB353 (Rat-401)</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuregulin 1 beta</td>
			<td>In house</td>
			<td>Made by S.L.C. (also available as 396-HB-050/CF from R&#38;D Systems)</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofibromin</td>
			<td>Santa Cruz Biotechnology&#160;</td>
			<td>sc-67</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice</td>
			<td>Jackson Laboratory</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat Dry Milk</td>
			<td>Walmart</td>
			<td>Great Value Brand</td>
			<td></td>
		</tr>
		<tr>
			<td>P0-GGF&#946;3 mice</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>Leica</td>
			<td>Paraplast 39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>PM-999</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (4%)</td>
			<td>Thermo Scientific</td>
			<td>J19943-K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount (Xylene Mounting Medium)</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toldedo</td>
			<td>Seven Excellence, 8603</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Dulbecco&#39;s)</td>
			<td>Corning</td>
			<td>20-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PMEL</td>
			<td>ABCAM&#160;</td>
			<td>EP4863(2), ab137078</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine Hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P5899-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Isoflurance Machine</td>
			<td>VetEquip Inhalation Anesthesia Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVA-DABCO (Aqueous Mounting Medium)</td>
			<td>Millipore Sigma</td>
			<td>10981100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Rice Cooker</td>
			<td>Beech Hamilton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S100B</td>
			<td>Agilent Dako&#160;</td>
			<td>Z0311&#160; (now GA504)</td>
			<td></td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>Ventana Medical Systems&#160;</td>
			<td>clone 1A4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate (Dihydrate)</td>
			<td>Fisher Scientific</td>
			<td>BP327-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox10</td>
			<td>ABCAM&#160;</td>
			<td>ab212843</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel histology mold</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>TCF4/TCFL2&#160;</td>
			<td>Cell Signaling&#160;</td>
			<td>(CH48H11) #2569</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>ACROS Organics</td>
			<td>348600050</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Corning</td>
			<td>25-051-31</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying, diagnosing, and grading malignant peripheral nerve sheath tumors in genetically engineered mouse models

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that subsequently transform into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Understanding the process by which a PN transforms into an MPNST would be facilitated by the availability of genetically engineered mouse (GEM) models that accurately replicate the PN-MPNST progression seen in humans with NF1. Unfortunately, GEM models with Nf1 ablation do not fully recapitulate this process. This led us to develop P0-GGF&#946;3 mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 (NRG1) in Schwann cells results in the development of PNs that progress to become MPNSTs with high frequency. However, to determine whether tumorigenesis and neoplastic progression in P0-GGF&#946;3 mice accurately model the processes seen in NF1 patients, we had to first prove that the pathology of P0-GGF&#946;3 peripheral nerve sheath tumors recapitulates the pathology of their human counterparts.
Here, we describe the specialized methodologies used to accurately diagnose and grade peripheral nervous system neoplasms in GEM models, using P0-GGF&#946;3 and P0-GGF&#946;3;Trp53+/- mice as an example. We describe the histologic, immunohistochemical, and histochemical methods used to diagnose PNs and MPNSTs, how to distinguish these neoplasms from other tumor types that mimic their pathology, and how to grade these neoplasms. We discuss the establishment of early-passage cultures from GEM MPNSTs, how to characterize these cultures using immunocytochemistry, and how to verify their tumorigenicity by establishing allografts. Collectively, these techniques characterize the pathology of PNs and MPNSTs that arise in GEM models and critically compare the pathology of these murine tumors to their human counterparts.

Author Spotlight: Genetically Engineered Mouse Models and Pathological Characterization of Neurofibromatosis Type 1 Associated Tumors

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models

Figure 2 illustrates examples of grossly evident neoplasms arising in P0-GGF&#946;3 mice. Tumors that are easily identifiable with the naked eye may be seen as masses distending body regions as shown in Figure 2A (arrow). When determining whether the neoplasm is potentially a peripheral nerve sheath tumor, it is essential to establish that the tumor is associated with a peripheral nerve. In this instance, an MRI scan (Figure 2B</s...

We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGF&#946;3 mouse model.

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that ...

identifying-diagnosing-grading-malignant-peripheral-nerve-sheath

Research

JoVE Journal

Cancer Research

731 Views.  Medical University of South Carolina. We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGFβ3 mouse model.

Video: Author Spotlight: Genetically Engineered Mouse Models and Pathological Characterization of Neurofibromatosis Type 1 Associated Tumors

Identifying Neurofibromas and Malignant Peripheral Nerve Sheath Tumors (MPNSTs) in P0-GGF&#946;3 Mice

Preparing Hematoxylin and Eosin-Stained Sections of Grossly Visible Tumors

Identifying Potential Plexiform Neurofibromas (PNs) and Malignant Peripheral Nerve Sheath Tumors (MPNSTs) by Performing Special Stains to Confirm Diagnoses