Expression of protein gene product 9.5 in epithelioid and conventional malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumor (MPNST) is a sarcoma of peripheral nerve origin, often associated with von Recklinghausen disease (neurofibromatosis), and rarely occurs as a complication of radiotherapy. (1) With the exception of cases arising in the context of von Recklinghausen disease, MPNST is often a diagnostic challenge because of its many histologic appearances and its lack of specific immunohistochemical markers. Since there is a frequent lack of S100 protein expression by MPNST, especially in the epithelioid variant, these tumors are difficult to diagnose without the aid of electron microscopy or the clinical history of neurofibromatosis.

Protein gene product 9.5 (PGP9.5), a broad neural marker, is expressed in nerve fibers and neurons of both the peripheral and central nervous systems. We compared the expression of PGP9.5 and S100 protein in 16 MPNSTs using immunohistochemistry on paraffin-embedded tissue.

MATERIALS AND METHODS

The consultation file of one of the authors (J.A.S.) and the surgical pathology file of the University of Texas Southwestern Medical Center, Dallas, Tex, were searched for MPNST from 1990 to 2001. The criteria for diagnosing MPNST were as follows: tumor origin in a nerve, presence of a contiguous neurofibroma, association with neurofibromatosis, electron microscopic findings, or by immunohistochemical exclusion. (2) Sixteen cases were identified, which formed the basis of this study. In addition, 6 monophasic synovial sarcomas, 9 leiomyosarcomas, and 5 dermatofibrosarcoma protuberans were included as controls.

Clinical histories were obtained from the patients' charts or letters from the referring pathologists.

Four-micrometer-thick sections were cut from the paraffin blocks and stained with hematoxylin-eosin. Additional paraffin sections of selected blocks were obtained for immunohistochemical studies, which were performed on an automated immunostainer (Ventana, Biotek System, Tucson, Ariz) with appropriate positive and negative controls run concurrently. Briefly, paraffin sections were mounted on charged glass slides, air-dried overnight, and then deparaffinized. To enhance the immunostaining, a heat-induced epitope-retrieval procedure was performed. After incubation with blocking serum, sections were incubated with primary antibodies (Table 1), followed by a biotinylated polyvalent secondary antibody solution. Sections were then incubated with horseradish peroxidase-conjugated avidin-biotin complex, followed by 3,3-diaminobenzidine and hydrogen peroxide.

For electron microscopy, tissue samples from 3 cases were post-fixed in 3% glutaraldehyde, and thin sections were stained with lead citrate and uranyl acetate. All grids were examined with a JEOL 100SX electron microscope (JEOL, Japan).

RESULTS

The clinicopathologic data for the 16 cases of MPNST are summarized in Table 2. The male-female ratio was 1: 3. The age range was from 20 to 77 years (median, 42 years; mean, 47 years). Three patients had neurofibromatosis. Sites of the tumor included the soft tissue of the head, orbit, chest wall, lung, mediastinum, abdomen, trunk, and extremities. The size of these tumors was available in 11 cases, which ranged from 3 to 18 cm (median, 8.0 cm; mean, 9.9 cm). Three cases (cases 7, 9, and 10) were recurrent tumors. The time interval between radiation therapy and the recurrent tumor was 3 years in 1 case of postradiation sarcoma.

Microscopic Findings

Ten cases were high-grade sarcomas, while the remaining 6 cases were of low grade. Of the high-grade tumors, 2 were of the epithelioid variant, 1 with rhabdomyomatous differentiation (malignant Triton tumor), 1 with osseous and chondroid differentiation, and 1 being a postradiation sarcoma. The remaining 5 high-grade sarcomas were of conventional type.

In the majority of the high-grade cases, spindle neoplastic cells were seen arranged in either sweeping fascicles, short intersecting fascicles, storiform pattern, or hemangiopericytomatous pattern (Figure 1). There was vascular proliferation with prominent perivascular cuffs of tumor cells. The irregular foci of necrosis showed a tendency to spare the tumor cells around blood vessels, resulting in a so-called peritheliomatous pattern in 3 cases. In other areas, geographic necrosis with peripheral palisade similar to that seen in glioblastoma multiforme was present (Figure 1). In 1 tumor (case 13), rounded or short fusiform nuclei aligned forming a palisade. Whorling of tumor cells reminiscent of tactoid differentiation and hyalinized nodules was noted in 5 cases. The stroma appeared focally myxoid in 6 cases. The nuclei were irregular, wavy, and elongated with occasional comma-shaped cells identified. The cytoplasmic borders were indistinct. Brisk mitotic activity was noted in all 10 cases. Rare multinucleated tumor giant cells were noted in 4 cases.

[FIGURE 1 OMITTED]

In 2 cases with epithelioid appearance (cases 5 and 6), a proliferation of atypical epithelioid cells with abundant cytoplasm, pleomorphic nuclei, and prominent nucleoli was seen (Figure 2). Nests and sheets of polygonal neoplastic cells with vacuolated eosinophilic cytoplasm were seen in 1 case, while short cords of large epithelioid cells arranged in a vaguely nodular pattern were seen in another. There was prominent peritheliomatous growth pattern and geographic necrosis. Numerous mitotic figures, greater than 6 per 10 high-power fields, were seen in both tumors.

[FIGURE 2 OMITTED]

Heterotopic osseous and chondroid elements were seen scattered throughout 1 tumor (case 3). Other areas showed myxoid hypocellular areas interspersed with densely cellular areas containing spindle-shaped cells arranged in alternating fascicles. One tumor (case 1) was a malignant Triton tumor, that is, an MPNST with rhabdomyomatous differentiation. In addition to a high-grade spindle cell proliferation, rhabdomyoblasts were seen scattered throughout the stroma. These large, rounded, or elongated desmin-positive rhabdomyoblasts had abundant eosinophilic cytoplasm with rarely identified cross-striations.

In the 6 low-grade cases, wavy spindle neoplastic cells with polymorphic nuclei were arranged in a vaguely herringbone pattern within a myxoid stroma. Occasional mitotic figures were noted. There was a coexisting neurofibromatous component in 1 case (case 12), which showed the typical histologic pattern of interlacing loose fascicles of cytologically benign spindle cells associated with a collagenous or myxoid stroma. A coexisting neurofibroma was not seen in the 2 primary tumors associated with neurofibromatosis.

Immunohistochemical Findings

The immunohistochemical results are summarized in Table 3. Immunoreactivity was scored semiquantitatively as follows: negative, 1+ for <5%, 2+ for 5% to 25%, 3+ for 25% to 50%, and 4+ for >50% of positive tumor cells. Both cytoplasmic and nuclear staining for PGP9.5 was observed with 1 to 4+ positivity in 15 cases (94%) (Figure 3). Ten cases, 2 epithelioid and 8 conventional MPNSTs, were reactive with PGP9.5 but negative for S100 protein. Eight of these cases exhibited 4 + staining intensity, and 1 case showed 3+ intensity.

[FIGURE 3 OMITTED]

In contrast, 6 cases were immunoreactive for S100 protein (38%). Five cases were immunoreactive for both S100 protein and PGP9.5. Staining with PGP9.5 was significantly stronger than that of S100 protein in 3 cases (cases 1, 9, and 11). Case 8 was unusual in that S100 protein was reactive in a greater number of neoplastic cells than PGP9.5. One case was negative for PGP9.5 but demonstrated 1+ S100 protein positivity (case 10).

Additional immunohistochemical studies were performed on the 16 MPNSTs in the initial workup to exclude the possibility of either monophasic synovial sarcoma or leiomyosarcoma (Table 3). Immunoreactivity in only rare cells was seen in 1, 2, and 2 cases for AE1/AE3, epithelial membrane antigen (EMA), and smooth muscle actin (SMA), respectively. None of the cases was positive for both AE1/AE3 and EMA. Both of the 2 cases (cases 5 and 6) with focal EMA positivity were of the epithelioid variant. Smooth muscle actin expression in case 1, a malignant Triton tumor, was seen predominantly in the rhabdomyoblasts.

Four of the 6 monophasic synovial sarcomas exhibited 3+ immunoreactivity to PGP9.5. Three of the 9 leiomyosarcomas demonstrated 3 to 4+ immunoreactivity. The anatomic sites of these cases were uterus, vulva, and stomach. Five dermatofibrosarcoma protuberans were negative for PGP9.5.

UItrastructural Findings

Electron microscopy was performed in 3 cases (cases 3, 4, and 5). All 3 cases had features of nerve sheath differentiation. Abundant basal lamina, interdigitating cell membrane, rough endoplasmic reticulum, and pinocytotic vesicles were seen in all 3 cases. Longitudinally oriented, cytoplasmic, intermediate filaments and whorled membranous structures resembling myelin bundles were present in 1 case (case 3).

COMMENT

The diagnostic challenge of MPNSTs resides mainly in distinguishing them from other spindle cell sarcomas, such as fibrosarcoma, monophasic synovial sarcoma, leiomyosarcoma, and neurotrophic/desmoplastic melanoma. Although S100 protein has been the marker of choice, its expression is focal and frequently negative in MPNST. Several studies have focused on the expression of other neural markers, such as myelin basic protein, Leu-7, and glial fibrillary acidic protein, in MPNST. Myelin basic protein and Leu-7 have been found to be less effective than S100 protein and are expressed in approximately 40% and 50% of MPNSTs, respectively. (3) Glial fibrillary acidic protein reactivity is lacking in the majority of MPNSTs. (4)

Protein gene product 9.5 was first detected in human brain extracts by high-resolution, 2-dimensional polyclonal polyacrylamide gel electrophoresis by Jackson and Thompson (5) in 1981. It is a 27 000-molecular weight protein whose function is not known. (5) Using polyclonal and monoclonal antibodies, Wilson et al (6) demonstrated that PGP9.5 is expressed in nerve fibers and neurons of both the central and peripheral nervous systems. This marker has subsequently been shown to be a broad marker for neuroectodermal-derived tumors. (7,8) In addition, part of the renal tubule, spermatogonia and Leydig cells of the testis, ova, theca externa, and theca interna of the corpus luteum of ovary also expressed PGP9.5. (6)

We found that PGP9.5 is a more sensitive marker than S100 protein for MPNSTs, with a sensitivity of 94% in comparison to 38% of S100 protein. There was strong positivity of this broad neural marker in contrast to the lack of S100 protein expression in 10 (62%) of our 16 cases. When there is coexpression of both markers, the degree and intensity of PGP9.5 immunoreactivity are either comparable to or greater than those of S100 protein. Four of our cases (cases 2, 3, 12, and 14) were negative for multiple markers in the initial workup, including cytokeratin, EMA, SMA, and S100 protein. The diagnosis of MPNST was made due to the clinical history of neurofibromatosis in 2 cases (cases 2 and 3), one of which had confirmatory electron microscopic findings. Of the remaining 2 cases, one case had a coexisting neurofibroma (case 12) and the other exhibited tactoid differentiation (case 14). All 4 cases demonstrated immunoreactivity with PGP9.5. It is in this setting that PGP9.5 would serve as a useful diagnostic adjunct.

With sophisticated antigen-retrieval techniques, no single immunohistochemical marker has remained specific for a certain entity. That is certainly the case with PGP9.5. Four of our 6 control cases of monophasic synovial sarcoma expressed PGP9.5; however, the degree of staining was less than that observed for MPNST. The majority of our MPNSTs demonstrated 4+ positivity with PGP9.5, in contrast to 3+ positivity in synovial sarcoma. This finding is in accordance with another study published in abstract form. (9) Although up to 95% of monophasic synovial sarcomas are immunoreactive for keratin markers or EMA, there is significant overlap between synovial sarcomas and MPNSTs in the immunophenotypic expression of S100 protein, EMA, and AE1/AE3. (10,11) A subgroup of MPNSTs expresses EMA and cytokeratin, and up to 60% of synovial sarcomas express S100 protein. (3,12-15) As previously described, both of our cases of epithelioid MPNST exhibited focal EMA positivity. (16) These findings in combination with our results again emphasize the limitation of immunohistochemistry and the importance of a broad immunohistochemical panel in the diagnostic workup of a spindle cell sarcoma.

Molecular studies have demonstrated the presence of the translocation t(X;18) in synovial sarcoma. This cytogenetic abnormality was considered distinctive for synovial sarcoma. (17) Recently, however, it was found in 75% of MPNSTs, showing that this cytogenetic abnormality is not specific for synovial sarcoma. (18) The overlap in histologic appearance, immunophenotype, and molecular changes between MPNST and synovial sarcoma complicates the differential diagnosis.

Three of our 9 control cases of leiomyosarcoma expressed PGP9.5. One case, being a uterine leiomyosarcoma, demonstrated strong PGP9.5 expression. Three leiomyomas expressed 4+ positivity in a study by Wang et al, (19) and PGP9.5 has been reported to express in smooth muscle of the myometrium. (19) However, leiomyosarcoma can usually be distinguished from MPNST by a combination of well-described histologic and immunohistochemical findings. The neoplastic cells have centrally located, blunt-ended nuclei and occasional juxtanuclear vacuoles. They are immunoreactive for SMA, muscle specific actin, and desmin.

Diffuse immunoreactivity with S100 protein argues against MPNST, a neoplasm in which the neoplastic cells typically stain focally. In a series of 16 metastatic malignant melanomas resembling MPNST, 14 tumors labeled strongly and diffusely for S100 protein. (20) In addition, a clinical history of a prior malignant melanoma or the presence of an in situ melanoma will aid in the differential diagnosis.

We have shown that although PGP9.5 is not a specific marker, it is a more sensitive marker than S100 protein for MPNST. When there is a lack of S100 protein expression and a broad panel of immunostains, including cytokeratin, EMA, and SMA, yields only focal or equivocal staining, PGP9.5 is a useful marker in confirming the neural origin of a spindle cell sarcoma.

Table 1. Immunohistochemical Stains

        Antibody                Clone      Dilution        Source

Protein gene product 9.5      3IA3          1:200     Biogenesis,
                                                        Brentwood, NH
S100                          Polyclonal    1:1200    Dako Corporation,
                                                        Carpinteria,
                                                        Calif
AE1/AE3                       AE1/AE3       1:80      Signet
                                                        Laboratories
                                                        Inc, Dedham,
                                                        Mass
Epithelial membrane antigen   E29           1:200     Dako
Smooth muscle actin           IA4           1:400     Dako
Desmin                        D33           1:50      Dako
Table 2. Clinicopathologic Features of 16 Cases of Malignant Peripheral
Nerve Sheath Tumor *

                        Case    Age,                              Size,
   Diagnostic Group      No.   y/Sex              Site             cm

History of neuro-         1     29/F   Anterior thigh mass         8.0
  fibromatosis
                          2     20/F   Right upper lobe of lung   18.0
History of neuro-         3     24/F   Sacral mass                 4.9
  fibromatosis and EM
  findings
EM findings               4     77/F   Right orbital soft          7.0
                                         tissue
EM findings and epi-      5     76/F   Left foot soft tissue       9.0
  thelioid variant
Epithelioid variant       6     38/F   Mediastinum                12.0
S100 positivity           7     57/M   Left calvarium              ...
                          8     32/F   Right lower leg mass       13.5
                          9     42/M   Soft tissue mass            ...
                         10     71/M   Right forearm               ...
                         11     67/F   Right chest wall            7.5
Histologic features      12     31/F   Left labia                  3.0
  of neural differen-    13     42/F   Right abdominal mass        ...
  tiation                14     52/F   Posterior mediastinum      10.0
                         15     36/F   Right thigh                 ...
                         16     52/M   Right foot mass             7.6

                        Case    Age,
   Diagnostic Group      No.   y/Sex        Clinical Presentation

History of neuro-         1     29/F   Neurofibromatosis
  fibromatosis
                          2     20/F   Neurofibromatosis; status post-
                                         resection of MPNST; now with
                                         metastatic tumor to lung
History of neuro-         3     24/F   Neurofibromatosis
  fibromatosis and EM
  findings
EM findings               4     77/F   Right orbital soft tissue mass
                                         involving periorbital bones,
                                         nasal mucosa, and upper and
                                         lower palpebral conjunctiva
EM findings and epi-      5     76/F   ...
  thelioid variant
Epithelioid variant       6     38/F   A large mediastinal mass
                                         extending into lung
S100 positivity           7     57/M   History of malignant fibrous
                                         histiocytoma 3 years prior;
                                         status postradiation therapy;
                                         now with recurrent tumor
                          8     32/F   ...
                          9     42/M   Recurrent tumor
                         10     71/M   Recurrent tumor
                         11     67/F   ...
Histologic features      12     31/F   ...
  of neural differen-    13     42/F   ...
  tiation                14     52/F   ...
                         15     36/F   ...
                         16     52/M   ...

* EM indicates electron microscopic; MPNST, malignant peripheral nerve
sheath tumor.
Table 3. Immunohistochemical Results of 16 Cases of
Malignant Peripheral Nerve Sheath Tumors *

Case
 No.   S100   PGP9.5   AE1/AE3   EMA   SMA

  1     2+      4+       --       --    1+
  2     --      2+       --       --    --
  3     --      4+       --       --    nd
  4     --      4+       --       --    nd
  5     --      4+       --       1+    nd
  6     --      4+       --       1+    --
  7     1+      1+       nd       nd    --
  8     3+      1+       --       --    --
  9     3+      4+       --       --    --
 10     1+      --       --       --    --
 11     1+      4+       --       --    --
 12     --      3+       --       --    --
 13     --      4+       --       --    nd
 14     --      4+       --       --    --
 15     --      4+       1+       --    --
 16     --      4+       --       --    1+

* PGP9.5 indicates protein gene product 9.5; EMA, epithelial membrane
antigen; SMA, smooth muscle actin; and nd, not done.

We thank Christa L. Hladik for her technical expertise.

References

(1.) Ducatman BS, Scheitheuer BW, Piepgras DG, Reiman HM, Ilstrup DM. Malignant peripheral nerve sheath tumors: a clinicopathotogic study of 120 cases. Cancer. 1986;57:2006-2021.

(2.) Enzinger FM, Weiss SW. Soft Tissue Tumors. 3rd ed. St Louis, Mo: CV Mosby; 1995:889-928.

(3.) Wick MR, Swanson PE, Scheithauer BW, Manivel JC. Malignant peripheral nerve sheath tumor: an immunohistochemical study of 62 cases. Am J Clin Pathol. 1987;87:425-433.

(4.) Memoli VA. Glial fibrillary acidic protein immunoreactivity in peripheral nerve sheath tumors. Ultrastruct Pathol. 1984;7:269-275.

(5.) Jackson P, Thompson RJ. The demonstration of new human brain-specific proteins by high resolution two-dimensional polyacrylamide gel electrophoresis. J Neurol Sci. 1981;49:429-438.

(6.) Wilson POG, Barber PC, Hamid QA, et al. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol. 1988;69:91-104.

(7.) Thompson RJ, Doran JF, Jackson P, Dhillon AP, Rode J. PGP9.5: a new marker for vertebrate neurons and neuroendocrine cells. Brain Res. 1983;278:224-228.

(8.) Rode J, Dhillon AP, Doran JF, Jackson P, Thompson RJ. PGP9.5, a new marker for human neuroendocrine tumors. Histopathology. 1985;9:147-158.

(9.) Wang AR, Weiss SW, Reed JA, Scott G. PGP9.5 and N-CAM are sensitive markers for malignant peripheral nerve sheath tumors (MPNSTs) [abstract]. Mod Pathol. 2000;13:16A.

(10.) Corson JM, Weiss LM, Banks-Schlegel SP, Pinkus GS. Keratin proteins and carcinoembryonic antigen in synovial sarcomas: an immunohistochemical study of 24 cases. Hum Pathol. 1984;15:615-621.

(11.) Fisher C. Synovial sarcoma: ultrastructural and immunohistochemical features of epithelial differentiation in monophasic and biphasic tumors. Hum Pathol. 1986;17:996-1008.

(12.) Guillou L, Wadden C, Kraus MD, Deitos AP, Fletcher CDM. S-100 protein reactivity in synovial sarcomas: a potentially frequent diagnostic pitfall: immunohistochemical analysis of 100 cases. Appl Immunohistochem. 1996;4:167-175.

(13.) Folpe AL, Schmidt RA, Chapman D, Gown AM. Poorly differentiated synovial sarcoma: immunohistochemical distinction from primitive neuroectodermal tumors and high-grade malignant peripheral nerve sheath tumors. Am J Surg Pathol. 1998;22:673-682.

(14.) Smith TA, Machen SK, Fisher C, Goldblum JR. Usefulness of cytokeratin subsets for distinguishing monophasic synovial sarcoma from malignant peripheral nerve sheath tumor. Am J Clin Pathol. 1999;112:641-648.

(15.) van de Rijn M, Barr FG, Xiong Q-B, Hedges M, Shipley J, Fisher C. Poorly differentiated synovial sarcoma: an analysis of clinical, pathologic, and molecular genetic features. Am J Surg Pathol. 1999;23:106-112.

(16.) Pinkus GS, Kurtin PJ. Epithelial membrane antigen: a diagnostic discriminant in surgical pathology: immunohistochemical profile in epithelial, mesenchymal, and hematopoietic neoplasms using paraffin sections and monoclonal antibodies. Hum Pathol. 1985;16:929-940.

(17.) Crew AJ, Clark J, Fisher C, et al. Fusion of SYT to two genes, SSX1 and SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma. EMBO J. 1995;14:2333-2340.

(18.) O'Sullivan MJ, Kyriakos M, Zhu X, et al. Malignant peripheral nerve sheath tumors with t(X;18): a pathologic and molecular genetic study. Mod Pathol. 2000; 13:1253-1263.

(19.) Wang AR, May D, Bourne P, Scott G. PGP9.5: a marker for cellular neurothekeoma. Am J Surg Pathol. 1999;23:1401-1407.

(20.) King R, Busam K, Rosai J. Metastatic malignant melanoma resembling malignant peripheral nerve sheath tumor: report of 16 cases. Am J Surg Pathol. 1999; 23:1499-1505.

Accepted for publication June 1, 2001.

From the Department of Pathology, The University of Texas Southwestern Medical Center, Dallas.

Reprints: Mai P. Hoang, MD, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9073 (e-mail: [e-mail]).