Neurofibroma are common benign peripheral nerve sheath tumors that occur sporadically as solitary nodules, or as multiple lesions in patients with NF1. Neurofibroma most commonly occur as cutaneous nodules, however, they may be found along any peripheral nerve, plexus or trunk. Malignant transformation occurs in approximately 5% of large plexiform lesions, but is rare in cutaneous and soft tissue neurofibroma. Malignant transformation is also more likely in patients with NF1 [1
]. Malignant peripheral nerve sheath tumors (MPNST) are uncommon, aggressive malignancies that arise within peripheral nerves and account for approximately 5% of soft tissue tumors. They may arise spontaneously; however, around 60% arise from neurofibroma, often of the plexiform type. Although rare in the general clinical population (incidence of 0.001%), MPNST arise in 8-13% of patients with neurofibromatosis 1[2
]. These tumors can be highly metastatic and often recur after resection and radiation therapy, often leading to death within 10 years of diagnosis. Due to this fact, MPNST are a major factor contributingto NF1 patient mortality [5
The putative tumor suppressor gene, p53, has been shown to play an important role in many malignancies [7
]. The p53 gene is located on the short arm of chromosome 17 and contains 393 amino acids. The wild type p53 represses abnormal cell proliferation and growth by acting at various cellular pathways. In recent years there has been additional evidence to suggest that genetic alterations at sites other than the NF1 gene may be important in the malignant transformation of NF1 neoplasms [9
]. A possible candidate in this process is p53, which has clearly been shown to play a role in various other cancers [14
CD44 is a major cell surface receptor for hyaluronic acid that is included in the group of cell adhesion molecules and is a membrane glycoprotein that is found in a wide variety of cell types. In lymphocytes, it functions as a homing receptor, but more importantly it is recognized for its interactions with various extracellular matrix components including hyaluronic acid. CD44 has multiple isoforms that are generated by alternative splicing of the mRNA, including or excluding certain exons. These variant exons (v3-v10) contribute to the molecule's ability to mediate significantly different functions, playing roles in cell-cell and cell-matrix adhesion and activation of high-affinity growth factor receptors. The extracellular matrix interactions of CD44 and its isoforms have also been shown to play a role in the growth of and infiltrative and metastatic behavior of various tumors [17
]. The standard form of CD44 is present on the surface of most human cells. Altered levels of CD44 expression have been detected in many types of human neoplasms [21
]. Thus detection of abnormal regulation of CD44 splicing could be helpful in diagnosis and prognostic evaluation of certain tumors as well as a possible diagnostic marker for theses neoplasms.
We have examined the expression of CD44 in benign and malignant neoplasms in NF1 patients to determine whether the differential expression of CD44, if any, correlates with infiltration or malignancy in these lesions. We have also undertaken an immunohistochemical assessment of the presence of functional wild type and mutant p53 protein in these tumors to elucidate whether or not p53 is also involved in the malignant transformation of neurofibroma.
Materials and methods
Twenty-eight benign and 16 malignant peripheral nerve sheath tumors were identified from the surgical pathology archives. Only specimens resected from patients with a known and documented clinical diagnosis of Neurofibromatosis Type I were selected for this study. Hematoxylin and eosin (H & E) stained sections along with any available special studies (S-100, etc.) were also reviewed to confirm the diagnosis. Forty-four tumors from 33 patients were selected and evaluated for immunoexpression of CD44, CD44v6 and p53.
p53: Mouse monoclonal antibody, DO-7 (Novocastra Laboratories Ltd) is specific for human p53 protein, wild type and mutant forms. Dilution used: 1:100. Immunopositivity with this antibody has been correlated with altered expression of the p53 gene and is an accepted method of detecting p53 abnormalities.
P53 immuno expression was semi-quantitated using Image ProPlus Image Analysis System (Media Cybernetics, Silver Spring, MD). Three areas with maximal immunoreactivity were sampled from each case. A medium power (20X) field was video-captured and analyzed for nuclear staining. Data is expressed as the number of positive nuclei/20X field and categorized as follows: 0–10%, 10–30%, 30–60% and greater than 60% immunopositive nuclei.
CD44H: Mouse, anti-human CD44 monoclonal antibody that recognizes human standard CD44 and all protein isoforms. It is unable to distinguish splice variants. Dilution used: 1:100. (R and D Systems).
CD44v6: Anti-human CD44 variant 6, mouse monoclonal antibody that recognizes any protein containing the variant 6 exon. Dilution used: 1:100. (R and D Systems).
All specimens had been fixed in 10% formalin and embedded in paraffin. Sections were incubated for 30 minutes in primary antibody. After multiple rinses in phosphate buffered saline, sections were incubated in a biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA), followed by a streptavidin-complex reagent containing horseradish peroxidase (Zymed Laboratories, Inc., South San Francisco, CA). Slides were rinsed and incubated with the chromogen DAB (3,3'-diaminobenzidine) and counterstained with methyl green or hematoxylin. Lymphoid tissue, normal epithelium (skin) and a known adenocarcinoma of the colon served as the positive controls for CD44, while the same tumor also served as a positive control for p53. Negative controls included either no primary antibody or nonspecific IgG applied to the sections.
Statistical analysis was performed with comparison between percentages of the individual groups using non para metric t-test as well as chisquared analysis. Statistical significance was defined as a p-value of less than 0.05.