Applied Immunohistochemistry & Molecular Morphology 10(2): 139-146, 2002
`
`© 2002 Lippincott Williams & Wilkins, Inc., Philadelphia
`
`Differential Expression of the PTEN Tumor
`Suppressor Protein in Fetal and Adult
`Neuroendocrine Tissues and Tumors: Progressive
`Loss of PTEN Expression in Poorly Differentiated
`Neuroendocrine Neoplasms
`
`*'l'Luoquan Wang, M.D.,
`
`'l'Ana Ignat, M.s., and *'l'Constantine A. Axiotis, M.D.
`
`Genetic alteration and loss of expression of tumor suppressor
`gene PTEN has been found in carcinomas of the breast, pros-
`tate, and endometrium, as well as in gliomas. PTEN expression
`in neural crest/neuroendocrine (NC/NE) tissues and in neo-
`plasms has not been reported. This study examines PTEN ex-
`pression in embryonal, fetal, and adult tissues by immunohis-
`tochemistry. The authors found high PTEN expression in em-
`bryonal, fetal, and adult NC/NE tissues. The authors also study
`the PTEN expression in NC/NE neoplasms (N = 37), includ-
`ing 5 melanocytic nevi, 2 melanomas, 9 carcinoids, 2 moder-
`ately differentiated neuroendocrine carcinomas, 13 poorly dif-
`ferentiated neuroendocrine carcinomas, 2 paragangliomas, 2
`pheochromocytomas, 2 medullary thyroid carcinomas, and 1
`neuroblastoma. All carcinoid tumors and melanocytic nevi
`showed moderate or strong immunostaining for PTEN. In con-
`trast, the majority of poorly differentiated neuroendocrine car-
`cinomas (7 of 13) were negative for PTEN (54%); the remain-
`der showed diminished reactivity. The two melanomas studied
`were also negative for PTEN immunostaining. The paragan-
`gliomas, pheochromocytomas, medullary thyroid carcinomas,
`and neuroblastoma all showed a strong PTEN stain. The au-
`thors postulate that PTEN is a differentiation marker for
`NC/NE tissue and tumors and that loss of PTEN expres-
`sion may represent an important step in the progression of
`NE tumors.
`Key Words: PTEN-tumor suppressor gene-
`Immunohistochemistry—Neuroendocrine—Carcinoid—
`Neuroendocrine carcinoma.
`
`AppliedImmurwhistochemistry & MolecularMarphology I0(2).-
`139-146, 2002.
`
`PTEN is a recently identified tumor suppressor gene
`mapped to chromosome TEN, having both lipid/tyrosine
`phosphatase activity and molecular homology to tensin
`(1,2). It is thought to play an important role in the fun-
`damental control of cell growth, death, adhesion, and
`migration (3-6). Transgenic mice, which harbor dis-
`
`From the *Department of Pathology, State University of New York,
`Brooklyn, New York; and TThe Kings County Hospital Center, Brook-
`lyn, New York.
`Address correspondence and reprint requests to Constantine A.
`Axiotis, M.D., State University of New York Health Science Center at
`Brooklyn, Box 25, 450 Clarkson Avenue, Brooklyn, NY 11202-2098.
`
`rupted PTEN, manifest early embryonic lethality,
`whereas heterologous mice that carry the PTEN+/- gene
`spontaneously develop tumors in Variety of organs, sug-
`gesting a dual role in ontogenesis and tumor suppression
`(7). Gerrnline mutations of PTEN have been detected in
`three human autosomal dominant disorders, Cowden dis-
`ease, L’Herrnitte-Duclos disease, and Barmayan-Zonana
`syndrome, which share some similar features including
`the formation of multiple benign tumors and an increased
`susceptibility to malignancy development (8-12). A high
`incidence of PTEN mutations has also been observed in
`
`including glioma, endometrial, and
`several tumors,
`breast carcinomas (13-19). Furthermore, loss of PTEN
`protein expression is frequent in high-grade breast and
`prostate carcinomas, suggesting that the loss of PTEN is
`a late event in the progression of these tumors (20-22).
`Recently, Gimm et al.(23) found high PTEN expres-
`sion in fetal and adult central and peripheral nervous
`systems. These data prompted us to investigate, using
`immunohistochemical methods, PTEN protein expres-
`sion in fetal and adult neural crest/neuroendocrine
`
`(NC/NE) tissues. We observed that PTEN protein was
`expressed in the fetal and adult pituitary glands, pancre-
`atic islets, adrenal medulla, peripheral nerve plexuses,
`and skin melanocytes. We subsequently investigated
`PTEN expression in a variety of NE neoplasms and
`found that progressive loss of PTEN protein expression
`occurred in poorly differentiated tumors.
`
`MATERIALS AND METHODS
`
`Tissues and Tumors
`
`The material studied comprised tissues from six em-
`bryos and fetuses (gestational age: 8, 11, 13, 17, 22, and
`25 weeks), two adults, and 37 NC/NE neoplasms (Table
`1) obtained from archival surgical and autopsy speci-
`mens in the Department of Pathology, Kings County
`Hospital Center, Brooklyn, New York. Blocks fixed in
`10% buffered formalin and embedded in paraffin were
`evaluated independently (by C.A.A. and L.W.) for ges-
`tational age, tissue type, and diagnosis. Immunostaining
`
`139
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`140
`
`L. WANG ET AL.
`
`TABLE 1. Grade and semiquantitative scoring of PTEN
`Immunostain in NC tumors
`
`Case
`
`Diagnosis
`
`Staining
`intensity
`
`Positive cells
`(%)
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`29
`30
`31
`32
`33
`34
`35
`36
`37
`
`Carcinoid, thymus
`Carcinoid, teratoma
`Carcinoid, colon
`Carcinoid, colon
`Carcinoid, colon
`Carcinoid, liver
`Carcinoid, mediastinum
`Carcinoid, appendix
`Carcinoid, small intestine
`MDNEC, kidney
`MDNEC, cen/ix
`PDNEC, endometrium
`PDNEC, gallbladder
`PDNEC, lung
`PDNEC, mediastinum
`PDNEC, lung
`PDNEC, lung
`PDNEC, lung
`PDNEC, cervix
`PDNEC, lung
`PDNEC, cervix
`PDNEC, lung
`PDNEC, cervix
`PDNEC, lung
`Pheochromacytoma
`Pheochromacytoma
`Paraganglioma
`Paraganglioma
`Medullary carcinoma, thyroid
`Medullary carcinoma, thyroid
`Neuroblastoma
`Melanocytic nevus, skin
`Melanocytic nevus, skin
`Melanocytic nevus, skin
`Melanocytic nevus, skin
`Melanocytic nevus, skin
`Melanoma, skin
`Melanoma, metastatic
`
`+++
`++
`+++
`+++
`+++
`+++
`++
`+++
`++
`++
`+
`++
`++
`++
`—
`+
`—
`—
`++
`—
`—
`—
`—
`+
`+++
`+++
`+++
`+++
`+++
`+++
`+++
`+++
`++
`+++
`++
`+++
`—
`—
`
`++++
`++++
`++++
`++++
`++++
`++++
`++++
`++++
`++++
`+++
`++
`+
`++
`+++
`O
`++
`O
`O
`+
`0
`O
`O
`0
`++
`+++
`++++
`++++
`+++
`++++
`+++
`+++
`++++
`+++
`++++
`++++
`+++
`0
`0
`
`MDNEC, moderately differentiated neuroendocrine carcinoma;
`PDNEC, poorly differentiated neuroendocrine carcinoma.
`
`was performed on 5-p. m sections from one or two rep-
`resentative areas. The diagnosis of neuroendocrine tu-
`mors was confirmed by synaptophysin and chromogran-
`in immunostaining. Immunostain for S-100 and HMB-45
`was performed on serial sections to confirm the recog-
`nition of olfactory cells, nerve plexuses, and melanocytes
`of skin, respectively.
`
`Classification of Neuroendocrine Tumors
`The neuroendocrine tumors were classified as carci-
`
`noid tumor, well-differentiated neuroendocrine carci-
`noma, moderately differentiated neuroendocrine carci-
`noma (MDNEC), and poorly differentiated neuroendo-
`crine carcinoma (PDNEC), according to Axiotis (24). A
`well-differentiated neuroendocrine carcinoma is equiva-
`lent to what the World Health Organization (1999) (25)
`classifies as an atypical carcinoid or to what Gould et al.
`(26) classifies as a well-differentiated neuroendocrine
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, No. 2, June 2002
`
`carcinoma grade I and II, and it is characterized by car-
`cinoidlike growth pattern with an apparent loss of archi-
`tectural organization (2 to 10 mitoses per high—power
`field and/or small foci of necrosis). Moderately differen-
`tiated neuroendocrine carcinoma is equivalent to atypical
`carcinoid (World Health Organization) or well-
`differentiated neuroendocrine carcinoma grade III
`(Gould et al.), and is it characterized by a carcinoidlike
`growth pattern with cellular discohesion and a prominent
`loss of architectural organization, 211 mitoses per 10
`high—power fields, and conspicuous areas of central ne-
`crosis. PDNEC is equivalent to small cell/large cell neu-
`roendocrine carcinoma (World Health Organization) or
`small cell/intermediate neuroendocrine carcinoma
`
`(Gould et al.), and it is characterized by the loss of car-
`cinoidlike growth pattern, numerous mitosis, and geo-
`graphic necrosis. PDNEC can be small cell type, large
`cell type, or mixed small/large cell type.
`
`Reagents
`A murine monoclonal antibody (sc-7974; Santa Cruz
`Biotech, Santa Cruz, CA) that recognizes the last
`100 C—terminal amino acids of human PTEN
`
`protein was used in all immunohistochemical analyzes.
`The specificity of this antibody has been shown in pre-
`vious studies (21,27).
`
`Immunostaining
`Immunostaining was performed on a Ventana Nexes
`IHC staining system by using the Nexus Enhanced DAB
`detection kit Cat#760—003 (Ventana, Tuscon, AZ) ac-
`cording to the manufacturer’s recommendations. Depar-
`affinized, rehydrated 4—p.m sections briefly underwent
`microwave antigen retrieval by using Antigen Retrieval
`Citra solution (BioGenex, San Ramone, CA). The pri-
`mary antibody against PTEN protein (1:50 dilution) was
`incubated at 37°C for 32 minutes. The melanin pigments
`from nevus, skin, or melanoma tissue sections were re-
`moved by standard treatment with 10% hydrogen perox-
`ide for 24-48 hours before histochemical immunostain-
`
`ing (28).
`
`Antigen retrieval
`Antigen retrieval techniques have dramatically im-
`proved the staining quality of archival tissue sections
`(29). An immunohistochemical study of PTEN protein
`expression in prostate carcinoma that used antigen re-
`trieval techniques by heating tissue sections at 100°C for
`10 minutes in a pH 6.0—citIate buffer in archival paraffin-
`embedded tissue sections has been successful (22). We
`optimized antigen retrieval conditions with a variety of
`formalin—fixed, paraffin—embedded archival tissues by
`heating tissue sections at 100°C for 5 minutes in a com-
`mercial citrate solution. A comparable staining intensity
`was achieved by using a commercial immunostaining kit
`and an automatic stainer.
`
`
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`PTEN TUMOR SUPPRESSOR PROTEIN EXPRESSION
`
`I41
`
`Controls
`
`Parallel sections incubated with PTEN-blocking
`peptide/PTEN antibody mixture determined the specific-
`ity of PTEN protein immunostaining. In detail, the anti-
`body against PTEN protein was preincubated with ex-
`cess PTEN-blocking peptide (Santa Cruz Biotech) and
`parallel slides were stained. Sections were also incubated
`with the immunoglobulin fraction of normal mouse se-
`rum (in place of the primary antibody) in the same pro-
`tein concentration as the primary antibody.
`
`Quantitation
`Cases with staining in more than 10% of cells were
`considered to have a positive result. PTEN immunostain-
`ing was serniquantitatively expressed as percentage of
`tumor cells stained <l0% (0), <25% (1+), <50% (2+),
`<75% (3+), >75% (4+); and intensity of immunostaining
`as weak staining (l+), moderate staining (2+), strong
`staining (3+).
`
`RESULTS
`
`Specificity of PTEN Protein Immunostaining
`Sections incubated with the anti—PTEN protein anti-
`body all showed diffuse, granular cytoplasmic immuno-
`staining with positive reactivity, except for a case of
`PDNEC that showed weak staining in both cytoplasm
`and nuclei. The parallel sections incubated with the anti-
`P'I‘EN/PTEN—blocking peptide mixture or the normal
`mouse serum did not show a reaction product (Fig. 1).
`
`PTEN Immunoreactivity in Fetal and Adult Tissues
`The strongest overall PTEN protein immunoreactivity
`was observed in the central and peripheral nervous sys-
`tems and in NC/NE tissues. The embryonal, fetal, and
`adult pituitary glands; pancreatic islets; the adrenal me-
`dulla; slcin melanocytes; the myenteric and mucosal gan-
`glia of gut; and the paravertebral ganglia stained strongly
`for PTEN protein (Figs. 2 and 3C). The nerve branches
`and twigs in peripheral organs and tissues were also
`strongly positive for PTEN.
`PTEN immunostaining in central nervous system
`(CNS) tissue showed differential expression during hu-
`man development. In early embryos, the CNS, including
`the retinal epithelium and primitive nasal olfactory cells,
`was diffusely and strongly positive for PTEN. Overall,
`the staining intensity decreases when the fetal CNS ma-
`tures. Adult brain and spinal cord tissues showed weakly
`positive staining for PTEN in both astrocytes and
`neurons (Fig. 3).
`In general, PTEN immunostaining results were nega-
`tive or weak in most nonneural tissues. Blood vessels
`
`were the only nonneural/neuroendocrine tissue that
`showed differential staining for PTEN product. In early
`fetal development,
`the aorta and small-vessel wall
`
`muscles tested positive for PTEN protein, whereas the
`adult aorta, medium-size muscular arteries, and small
`vessels and capillaries lost PTEN protein expression.
`Adult thyroid glands, breast tissue, basal layers of skin,
`the esophagus, and the prostate show focal and weak
`PTEN protein staining.
`
`PTEN Protein Immunoreactivity in NCINE Tumors
`The 37 NC/NE tumors listed in Table 1 were evalu-
`
`ated for PTEN protein immunoreactivity. Figures 4 and 5
`are representations of the results obtained. All carcinoid
`tumors, melanocytic nevi, paragangliomas, pheochromo-
`cytomas, medullary thyroid carcinomas, and neuroblas-
`tomas showed moderate or strong immunostaining for
`PTEN protein, and a majority of the tumor cells
`had positive results (>75%). However, 7 of 13 (54%)
`PDNECs were negative for PTEN immunostaining; the
`remaining PDNECs partially lost PTEN expression.
`Moderately differentiated neuroendocrine carcinomas
`showed a weak or a moderate PTEN protein stain.
`The two melanomas examined were also negative for
`PTEN product.
`
`DISCUSSION
`
`The present immunohistochemical data show 1) dif-
`ferential PTEN expression during CNS development; 2)
`marked PTEN expression in both fetal and adult NC/NE
`tissues; and 3) differential PTEN expression in well-
`differentiated NE tumors and poorly differentiated NE
`tumors. Because PTEN is not a structural protein, we
`believe that its expression is important for the differen-
`tiation of CNS and NC/NE tissues. PTEN inactivation in
`
`a transgenic mouse model results in early embryonic
`lethality. PTEN+/- and Chimeric mice showed suscepti-
`bility to subsequent tumor development, suggesting a
`dual role in both ontogenic development and tumor sup-
`pression (7). However, the formation and development
`of CNS and NC/NE tissues were not specifically exam-
`ined in this PTEN knockout animal model. It would be
`
`reasonable to speculate that the development of the ner-
`vous and vascular systems is mostly likely affected be-
`cause of the high PTEN expression in these two systems.
`In addition, Cowden’s disease, L’Hermitte—Duclos dis-
`ease, and Barmayan-Zonana syndrome are characterized
`by developmental defects such as vascular malforma-
`tions and features such as dysplastic gangliocytoma of
`the cerebellum and mental retardation, further suggesting
`that PTEN plays a role in both neural and vascular
`development (10-12).
`Molecular characterization of PTEN product has
`shown a high degree of homology to lipid/protein tyro-
`sine phosphatases and tensin (1,30). The lipid phospha-
`tase activity of PTEN product is related to the inhibition
`of cell cycle progression and G1 arrest through regula-
`tion of the PI3K/Akt pathway (31). Akt also is one of the
`
`
`
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`142
`
`L. WANG ET AL.
`
`" .:-_
`~'.
`"-_ ' .,$s.'f¢'-
`
`FIG. 1. A: Carcinoid incubated with anti-PTEN monoclonal an-
`tibody shows strong PTEN reactivity. B: The parallel section
`incubated with anti-PTEN antibody/blocking peptide mixture
`shows specific blocking of PTEN immunoreactivity.
`
`FIG. 2. A: Pancreatic islet. B: Ad-
`enohypophysis. C: Nerve plexus of
`the intestine. D: Olfactory cells of na-
`sal mucosa. E: Adrenal medulla. F:
`Skin melanocytes. All show PTEN im-
`
`.;u-
`
`munoreactivity.
`show weak PTEN staining.
`
`-'
`" "
`
`.
`
`'
`
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`
`.
`
`'
`'
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`-
`
`FIG. 3. A and 0: Embryonic brain and spi-
`nal cord cells (11-week gestational age)
`show strong PTEN immunoreactivity. B
`and D: Adult brain and spinal cord cells
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, No. 2, June 2002
`
`Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
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`

`PTEN TUMOR SUPPRESSOR PROTEIN EXPRESSION
`
`I43
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`FIG. 4. Positive PTEN immunostaining. A: Neuroblastoma. B: Pheochromocytoma. C: Paraganglioma. D: Melanocytic
`nevus. E: Thyroid medullary carcinoma.
`
`58
`
`_:
`
`
`
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`ity. B: Moderately differentiated neuroendocrine carci-
`noma shows moderate PTEN immunoreactivity. C: Poorly
`differentiated neuroendocrine carcinoma shows weak fo-
`cal PTEN immunoreactivity.
`
`key regulatory molecules involved in the protection of
`cells against apoptosis (32-34). Tensin binds actin fila-
`ments at focal cellular adhesions,
`integrin-containing
`complexes, focal adhesion kinase, and growth factor re-
`ceptors, all of which have been implicated in cell growth
`regulation, cell mobility, cell—stromal interaction, and an-
`giogenesis (30,35). Because these events are key ele-
`ments in both tissue developmental differentiation and
`tumorigenesis, it is not surprising that the highly homolo-
`gous PTEN protein molecule may play a dual role in
`both human development and tumor suppression. Actu-
`ally, a similar characteristic has been found recently in
`another important molecule for the development of neu-
`ral crest derivatives, which is encoded by c-ret proto-
`oncogene (36). It belongs to the membrane receptors of
`the protein tyrosine kinase family and is expressed dur-
`ing mouse development in a number of tissues, including
`neural crest derivatives such as dorsal root ganglia, sym-
`pathetic and enteric ganglia, and neuroretinal and migrat-
`ing neural crest cells at earlier stages (37). High levels of
`c-ret transcripts have been found in pheochromocytomas
`and medullary thyroid carcinomas, which may be asso-
`ciated with a syndrome called multiple endocrine neo-
`plasia (38). The involvement of c-ret in these tumors has
`been supported by the identification of mutations in the
`ret gene. These mutations probably generate dominant
`tyrosine kinase—activated receptors. Mutations of c-ret
`have also been found in patients with Hirschsprung’s
`disease, which results from a congenital deficiency in the
`innervation of the posterior gut (39). The nature of the
`t ti
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`mu 3 ‘ms 15
`ere” 1“ cancer Syn Omes an 1“
`“Sc '
`Sphlhg diseases ih Which deletions ih the Tet gehe Suggest
`a loss of function.
`
`Copyright © Lippiricott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`West-Ward Pharm.
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`
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`

`144
`
`L. WANG ET AL.
`
`PTEN is highly expressed in peripheral nerve ganglia,
`adrenal medulla, pancreatic islets, pituitary glands, and
`melanocytes. PTEN staining was not detected in the gas-
`trointestinal and respiratory diffuse neuroendocrine sys-
`tem. The ontogeny of the diffuse neuroendocrine system
`is still controversial (24). Some studies favor its deriva-
`tion from endoderrnal stem cells, whereas others support
`the theory of migration from the neural crest. The NC
`stem cells have broad potential in the formation of dif-
`ferent tissues, from cartilage to melanocytes. The step-
`wise restrictions of pluripotent progenitors may result
`from the expressional regulation of different genes (40,
`41). Therefore, it is not surprising that the NC-derived
`cells do not express PTEN equally, just as chromogranin
`and synaptophysin are expressed in most NE organs but
`not in melanocytes (42). In addition, we found no PTEN
`immunostaining in either the parathyroid gland or its
`adenoma. The classification of the parathyroid gland as a
`NE organ has been questioned, although positive immu-
`nostaining for synaptophysin has been reported (43).
`The regulation of PTEN expression was not well un-
`derstood until recently when a p53-binding element up-
`stream of the PTEN gene was identified (44). Deletion
`and mutation analyzes showed that this element is nec-
`essary for inducible transactivation of PTEN by p53. As
`a tumor suppressor, p53 also plays a role in the regula-
`tion of cell proliferation and apoptosis. It would be in-
`teresting to know how these two tumor suppressors in-
`teract in the regulation of cell proliferation and death. A
`p53—independent element controlling constitutive expres-
`sion of PTEN was also identified, indicating the com-
`plexity of PTEN expression regulation (44).
`PTEN is highly expressed in benign and low-grade NE
`tumors. However, most PDNECs completely lack or par-
`tially lose PTEN expression. Loss of PTEN expression is
`also commonly seen in high-grade prostate and breast
`carcinomas. PTEN expression is even elevated in benign
`and low-grade tumors of the prostate and the breast, such
`as prostatic intraepithelial neoplasia and breast fibroad-
`enoma (20,2l,27,45,46). Similarly, the genetic alteration
`of PTEN is frequent in high-grade glioma but not in
`low-grade glioma (14,47). These data suggest that loss of
`the tumor suppressor PTEN product expression is a late
`event in the progression of these types of tumor. Because
`the PTEN product functions as a tumor suppressor, loss
`of or reduction of expression in NE tumors may be a
`meaningful prognostic marker. Noticeably, the mutation
`of another tumor suppressor gene, p53, is also considered
`a late event in most types of carcinoma (48,49). Tumor
`suppressor genes, such as p53 and the retinoblastoma
`gene product, have been found in NE neoplasms. The
`p53 gene is progressively overexpressed in well-
`differentiated neuroendocrine carcinoma to PDNEC,
`whereas the retinoblastoma gene is inactivated only in
`PDNEC (50,51). It is also noted that PTEN is highly
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, N0. 2, June 2002
`
`expressed in nevus, whereas its expression in two cases
`of melanoma is lost.
`
`The consistent high expression of PTEN protein in
`fetal and adult NC/NE tissues and in differentiated
`NC/NE tumors indicates that PTEN is a NC/NE differ-
`
`entiation marker. PTEN product expression was partially
`or completely lost in PDNEC. It is noted that some
`PDNEC only show weak, focal, or even negative immu-
`nostain for synaptophysin and/or chromogranin, the two
`most commonly used NE differentiation markers. We
`saw two cases of PDNEC that were negative for synap-
`tophysin stain and showed stronger staining for PTEN
`than for chromogranin. Therefore, we propose P'I‘EN
`protein immunostain can be used as an adjunct immuno-
`histochemical marker in the diagnosis of NC/NE tumors.
`PDNECs are among the most aggressive human neo-
`plasms. Patients often have a dismal prognosis, and a
`better treatment regimen is certainly desired. PTEN lipid
`phosphatase activity acts to break cell cycle progression
`by inhibiting the PI3K pathway (52-54). Activated forms
`of proto-oncogene Akt were capable of overriding a
`PTEN-mediated cell cycle block, raising the possibility
`that Akt might be an important downstream target of
`PTEN with respect to cell cycle progression (33,55). It
`has also been shown that cell lines and tumors in which
`
`PTEN is lost have elevated levels of activated Akt (56-
`59). Thus, the loss of immunohistochemical detection of
`PTEN might predict the presence of activated Akt and, in
`turn, might become useful as a factor predicting success
`of therapies directed against this pathway. We also pro-
`pose that PTEN may be used as a pharmacodiagnostic
`and prognostic marker.
`Although we did not assess the genetic status of PTEN
`in NE neoplasms, the loss of PTEN protein expression,
`as assessed by immunohistochemistry, may reflect the
`majority of the possible mechanisms that result in PTEN
`inactivation. These would include direct inactivation by
`homozygous deletion, nonsense mutation, certain inter-
`nal deletions, and promotor methylation, or indirect in-
`activation such as loss of a PTEN-directed transcription
`factor or posttranscriptional modification. Some mis-
`sense mutations, which do not grossly destabilize the
`protein product, would not be accounted for by immu-
`nohistochemistry. Therefore, we believe immunohisto-
`chemistry is the optimal method for evaluating the func-
`tional status of PTEN because it would detect a loss of
`
`PTEN function induced by a majority of the mechanisms
`through which gene products are inactivated.
`
`REFERENCES
`
`1. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine
`phosphatase gene mutated in human brain, breast, and prostate
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