`
`© 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
`
`*†Luoquan Wang, M.D., †Ana Ignat, M.S., and *†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.
`K e y W o r d s : P T E N - t u m o r
`s u p p r e s s o r g e n e —
`Immunohistochemistry—Neuroendocrine—Carcinoid—
`Neuroendocrine carcinoma.
`
`AppliedImmunohistochemistry&MolecularMorphology 10(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 †The 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). Germline mutations of PTEN have been detected in
`three human autosomal dominant disorders, Cowden dis-
`ease, L’Hermitte-Duclos disease, and Bannayan-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
`several tumors, including glioma, endometrial, and
`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
`
`ARGENTUM
`Exhibit 1051
`
`000001
`
`
`
`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, cervix
`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
`
`+++
`++
`+++
`+++
`+++
`+++
`++
`+++
`++
`++
`+
`++
`++
`++
`—
`+
`—
`—
`++
`—
`—
`—
`—
`+
`+++
`+++
`+++
`+++
`+++
`+++
`+++
`+++
`++
`+++
`++
`+++
`—
`—
`
`++++
`++++
`++++
`++++
`++++
`++++
`++++
`++++
`++++
`+++
`++
`+
`++
`+++
`0
`++
`0
`0
`+
`0
`0
`0
`0
`++
`+++
`++++
`++++
`+++
`++++
`+++
`+++
`++++
`+++
`++++
`++++
`+++
`0
`0
`
`MDNEC, moderately differentiated neuroendocrine carcinoma;
`PDNEC, poorly differentiated neuroendocrine carcinoma.
`
`was performed on 5- 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, ⱖ11 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-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-citrate 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.
`
`000002
`
`
`
`PTEN TUMOR SUPPRESSOR PROTEIN EXPRESSION
`
`141
`
`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 semiquantitatively expressed as percentage of
`tumor cells stained <10% (0), <25% (1+), <50% (2+),
`<75% (3+), >75% (4+); and intensity of immunostaining
`as weak staining (1+), 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-
`PTEN/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; skin 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 NC/NE 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 Bannayan-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
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, No. 2, June 2002
`
`000003
`
`
`
`142
`
`L. WANG ET AL.
`
`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-
`munoreactivity.
`
`FIG. 3. A and C: Embryonic brain and spi-
`nal cord cells (11-week gestational age)
`show strong PTEN immunoreactivity. B
`and D: Adult brain and spinal cord cells
`show weak PTEN staining.
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, No. 2, June 2002
`
`000004
`
`
`
`PTEN TUMOR SUPPRESSOR PROTEIN EXPRESSION
`
`143
`
`FIG. 4. Positive PTEN immunostaining. A: Neuroblastoma. B: Pheochromocytoma. C: Paraganglioma. D: Melanocytic
`nevus. E: Thyroid medullary carcinoma.
`
`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
`mutations is different in cancer syndromes and in Hirsch-
`sprung disease, in which deletions in the ret gene suggest
`a loss of function.
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, No. 2, June 2002
`
`FIG. 5. A: Carcinoid shows strong PTEN immunoreactiv-
`ity. B: Moderately differentiated neuroendocrine carci-
`noma shows moderate PTEN immunoreactivity. C: Poorly
`differentiated neuroendocrine carcinoma shows weak fo-
`cal PTEN immunoreactivity.
`
`000005
`
`
`
`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 endodermal 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 (20,21,27,45,46). 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, No. 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 PTEN
`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.
`䊐
`
`REFERENCES
`
`1. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine
`phosphatase gene mutated in human brain, breast, and prostate
`cancer [see comments]. Science 1997;275:1943–7.
`2. Steck PA, Pershouse MA, Jasser SA, et al. Identification of a
`candidate tumor suppressor gene, MMAC1, at chromosome
`
`000006
`
`
`
`PTEN TUMOR SUPPRESSOR PROTEIN EXPRESSION
`
`145
`
`10q23.3 that is mutated in multiple advanced cancers. Nat Genet
`1997;15:356–62.
`3. Huang H, Potter CJ, Tao W, et al. PTEN affects cell size, cell
`proliferation and apoptosis during Drosophila eye development.
`Development 1999;126:5365–72.
`4. Lu Y, Lin YZ, LaPushin R, et al. The PTEN/MMAC1/TEP tumor
`suppressor gene decreases cell growth and induces apoptosis and
`anoikis in breast cancer cells. Oncogene 1999;18:7034–45.
`5. Paramio JM, Navarro M, Segrelles C, et al. PTEN tumor suppres-
`sor is linked to the cell cycle control through the retinoblastoma
`protein. Oncogene 1999;18:7462–8.
`6. Tian XX, Pang JC, To SS, et al. Restoration of wild-type PTEN
`expression leads to apoptosis, induces differentiation, and reduces
`telomerase activity in human glioma cells. J Neuropathol Exp Neu-
`rol 1999;58:472–9.
`7. Di Cristofano A, Pesce B, Cordon-Cardo C, et al. PTEN is essen-
`tial for embryonic development and tumor suppression. Nat Genet
`1998;19:348–55.
`8. Liaw D, Marsh DJ, Li J, et al. Germline mutations of the PTEN
`gene in Cowden disease, an inherited breast and thyroid cancer
`syndrome. Nat Genet 1997;16:64–7.
`9. Marsh DJ, Dahia PL, Zheng Z, et al. Germline mutations in PTEN
`are present in Bannayan-Zonana syndrome [letter]. Nat Genet
`1997;16:333–4.
`10. Bannayan GA. Lipomatosis, angiomatosis, and macrencephalia. A
`previously undescribed congenital syndrome. Arch Pathol 1971;
`92:1–5.
`11. Zonana J, Rimoin DL, Davis DC. Macrocephaly with multiple
`lipomas and hemangiomas. J Pediatr 1976;89:600–3.
`12. Eng C. Cowden syndrome and LHérmitte-Duclos disease in a fam-
`ily: a single genetic syndrome with pleiotropy. J Med Genet 1994;
`31:458–61.
`13. Matias-Guiu X, Catasus L, Bussaglia E, et al. Molecular pathology
`of endometrial hyperplasia and carcinoma. Hum Pathol 2001;32:
`569–77.
`14. Davies MP, Gibbs FE, Halliwell N, et al. Mutation in the
`PTEN/MMAC1 gene in archival low grade and high grade glio-
`mas. Br J Cancer 1999;79:1542–8.
`15. Duerr EM, Rollbrocker B, Hayashi Y, et al. PTEN mutations in
`gliomas and glioneuronal tumors. Oncogene 1998;16:2259–64.
`16. Bussaglia E, del Rio E, Matias-Guiu X, et al. PTEN mutations in
`endometrial carcinomas: a molecular and clinicopathologic analy-
`sis of 38 cases. Hum Pathol 2000;31:312–7.
`17. Levine RL, Cargile CB, Blazes MS, et al. PTEN mutations and
`microsatellite instability in complex atypical hyperplasia, a precur-
`sor lesion to uterine endometrioid carcinoma. Cancer Res 1998;
`58:3254–8.
`18. Chen J, Lindblom P, Lindblom A. A study of the PTEN/MMAC1
`gene in 136 breast cancer families. Hum Genet 1998;102:124–5.
`19. Chen ST, Yu SY, Tsai M, et al. Mutation analysis of the putative
`tumor suppression gene PTEN/MMAC1 in sporadic breast cancer.
`Breast Cancer Res Treat 1999;55:85–9.
`20. Whang YE, Wu X, Suzuki H, et al. Inactivation of the tumor
`suppressor PTEN/MMAC1 in advanced human prostate cancer
`through loss of expression. Proc Natl Acad Sci U S A1998;95:
`5246–50.
`21. Perren A, Weng LP, Boag AH, et al. Immunohistochemical evi-
`dence of loss of PTEN expression in primary ductal adenocarci-
`nomas of the breast. Am J Pathol 1999;155:1253–60.
`22. McMenamin ME, Soung P, Perera S, et al. Loss of PTEN expres-
`sion in paraffin-embedded primary prostate cancer correlates with
`high Gleason score and advanced stage. Cancer Res 1999;59:
`4291–6.
`23. Gimm O, Attie-Bitach T, Lees JA, et al. Expression of the PTEN
`tumor suppressor protein during human development. Hum Mol
`Genet 2000;9:1633–9.
`24. Axiotis CA. The neuroendocrine lung. In: Livolsi V, Asa A, eds.
`Endocrine Pathology. Philadelphia: Harcourt, 2002:261–296.
`25. World Health Organization. Histological typing of lung and pleural
`tumors. In: Sobin L, ed. International Histologic Classification of
`Tumors. Geneva, Switzerland: World Health Organization;
`1999:1–156.
`26. Warren W, Memoli V, Gould V. Well differentiated and small cell
`
`neuroendocrine carcinomas of the lung. Two related but distinct
`clinicopathologic entities. Virchows Arch B Cell Pathol 1988;55:
`299–310.
`27. Depowski P, Rosenthal S, Ross J. Loss of expression of the PTEN
`gene protein product is associated with poor outcome in breast
`cancer. Mod Pathol 2001;14:672–6.
`28. Putt FA. Manual of Histopathologic Staining Methods. New York,
`John Wiley & Sons, 1972.
`29. Shi S, Gu J, Jurrens J, Cote R, Taylor C. Development of the
`antigen retrieval technique: Philosophical and theoretical bases. In:
`Shi S, Gu J, Taylor C, eds. Antigen retrieval techniques: Eaton
`Publishing, 2000:17–39.
`30. Chen H, Ishii A, Wong WK, Chen LB, Lo SH. Molecular charac-
`terization of human tensin. Biochem J 2000;351 Pt 2:403–11.
`31. Stambolic V, Suzuki A, de la Pompa JL, et al. Negative regulation
`of PKB/Akt-dependent cell survival by the tumor suppressor
`PTEN. Cell 1998;95:29–39.
`32. Davies MA, Koul D, Dhesi H, et al. Regulation of Akt/PKB ac-
`tivity, cellular growth, and apoptosis in prostate carcinoma cells by
`MMAC/PTEN. Cancer Res 1999;59:2551–6.
`33. Li J, Simpson L, Takahashi M, et al. The PTEN/MMAC1 tumor
`suppressor induces cell death that is rescued by the AKT/protein
`kinase B oncogene. Cancer Res 1998;58:5667–72.
`34. Yuan ZQ, Sun M, Feldman RI, et al. Frequent activation of AKT2
`and induction of apoptosis by inhibition of phosphoinositide-3-OH
`kinase/Akt pathway in human ovarian cancer. Oncogene 2000;19:
`2324–30.
`35. Soussi T. PTEN (MMAC, TEP-1): phosphatase and tensin homo-
`log deleted on chromosome 10. Bull Cancer 1999;86:715–6.
`36. Douarin NM, Dupin E, Ziller C. Genetic and epigenetic control in
`neural crest development. Curr Opin Genet Dev 1994;4:685–95.
`37. Pachnis V, Mankoo B, Costantini F. Expression of the c-net proto-
`oncogene during mouse embryogenesis. Development 1993;119:
`1005–17.
`38. Mulligan LM, Kwok JBJ, Healey CS, et al. Germ-line mutations of
`the RET proto-oncogene in multiple endocine neoplasia type 2A.
`Nature 1993;363:458–60.
`39. Edery P, Lyonnet S, Mulligan LM, et al. Mutations of the RET
`Proto-oncogene in Hirschsprung’s disease. Nature 1994;367:
`378–80.
`40. Bronner-Fraser M. Neural crest cell formation and migration in the
`developing embryo. FASEB J 1994;8:699–707.
`41. Vaglia J, Hall BK. Regulation of neural crest cell populations:
`occurrence, distribution and underlying mechanisms. Int J Dev
`Biol 1999;43:95–110.
`42. Gould VE, Wiedemann B, Lee I, et al. Synaptophysin expression
`in neuroendocrine neoplasms as determined by immunocytochem-
`istry. Am J Pathol 1986;126:243–57.
`43. Gould VE, Memoli V, Chejfec G, Johannessen JV. The APUD cell
`system and its neoplasms/Observation on the significance and
`limitations of the concept. Surg Clin North Am 1979;59:93–108.
`44. Stambolic V, MacPherson D, Sas D, et al. Regulation of PTEN
`transcription by p53. Mol Cell 2001;8:317–25.
`45. Bose S, Wang SI, Terry MB, et al. Allelic loss of chromosome
`10q23 is associated with tumor progression in breast carcinomas.
`Oncogene 1998;17:123–7.
`46. Garcia JM, Silva JM, Dominguez G, et al. Allelic loss of the PTEN
`region (10q23) in breast carcinomas of poor pathophenotype.
`Breast Cancer Res Treat 1999;57:237–43.
`47. Rasheed BK, Stenzel TT, McLendon RE, et al. PTEN gene muta-
`tions are seen in high-grade but not in low-grade gliomas. Cancer
`Res 1997;57:4187–90.
`48. Wang L, Yu Y, Sell S. P53 and PCNA as differentiation markers
`of hepatocellular carcinomas. J Tumor Marker Oncol 1998;13:
`5–13.
`49. Hillstein MC, Sidransky D, Harris CC. P53 mutations in human
`cancers. Science 1991;253:49–53.
`50. Barbareschi M, Girlando S, Mauri FA, et al. Tumor suppressor
`gene products, proliferation, and differentiation markers in lung
`neuroendocrine neoplasms. J Pathol 1992;166:343–50.
`51. Harbor JW, Lai SL, Whang-Peng J, et al. Abnormalities in struc-
`ture and expression of the human retinoblastoma gene in SCLC.
`Science 1997;241:353–7.
`
`Applied Immunohistochemistry & Molecular Morphology, Vol. 10, No. 2, June 2002
`
`000007
`
`
`
`146
`
`L. WANG ET AL.
`
`52. Hlobilkova A, Guldberg P, Thullberg M, et al. Cell cycle arrest by
`the PTEN tumor suppressor is target cell specific and may require
`protein phosphatase activity. Exp Cell Res 2000;256:571–7.
`53. Maehama T, Dixon JE. PTEN: a tumor suppressor that functions as
`a phospholipid phosphatase. Trends Cell Biol 1999;9:125–8.
`54. Myers MP, Pass I, Batty IH, et al. The lipid phosphatase activity of
`PTEN is critical for its tumor supressor function. Proc Natl Acad
`Sci U S A 1998;95:13513–8.
`55. Sun H, Lesche R, Li DM, et al. PTEN modulates cell cycle pro-
`gression and cell survival by regulating phosphatidylinositol
`3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.
`Proc Natl Acad Sci U S A 1999;96:6199–204.
`56. Dah