An Analysis of Potential Surrogate Markers of Target-Specific Therapy in Archival Materials of Adrenocortical Carcinoma
Megumi Nakamura · Yasuhiro Miki · Jun-ichi Akahira · Ryo Morimoto . Fumitoshi Satoh . Shigeto Ishidoya ·
Yoichi Arai . Takashi Suzuki . Yutaka Hayashi · Hironobu Sasano
Published online: 28 January 2009 C Humana Press Inc. 2009
Abstract Adrenocortical carcinoma (ACC) is a rare neo- plasm but some of the cases are highly malignant. Clinical outcome of the patients with advanced ACC still remained
M. Nakamura · Y. Miki · J .- i. Akahira · H. Sasano Department of Pathology, Graduate School of Medicine, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan
M. Nakamura · Y. Hayashi Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan
R. Morimoto · F. Satoh
Division of Nephrology, Endocrinology and Vascular Medicine, Department of Internal Medicine, Graduate School of Medicine, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan
S. Ishidoya · Y. Arai Department of Urology, Graduate School of Medicine, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan
T. Suzuki Department of Pathology, School of Health Science, Tohoku University, 2-1 Seiryo-machi, Aobaku, Sendai, Miyagi 980-8575, Japan
H. Sasano ☒ Department of Pathology, School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai-shi, Miyagi 980-8574, Japan e-mail: hsasano@patholo2.med.tohoku.ac.jp
poor or dismal despite recent development of aggressive antitumor therapies. Target-specific therapies have been developed in a number of human malignancies and resulted in therapeutic benefits in some cancer patients. However, these therapies are only effective in the cases in which corresponding targets are expressed in tumor tissues. Therefore, we evaluated expression of potential surrogate markers using immunohistochemistry in archival materials of adrenocortical carcinoma in order to explore the potential application of target specific therapies in ACC in this study. We immunolocalized ten established or potential surrogate markers of target-specific therapies, located in the Ras/ extracellular signal-regulated kinase and phosphatidylinosi- tol-3 kinase/Akt pathways, in 41 ACC cases, 54 adreno- cortical adenoma (ACA) cases, and five nonpathological adrenal glands and correlated the findings with clinicopath- ological factors of the patients. Among these markers examined, only epidermal growth factor receptor (EGFR) was significantly more abundant in ACC than in ACA (P< 0.01). These findings suggest that the agents which specifically inhibit signal transductions through EGFR such as monoclonal antibodies against EGFR are considered to be worthwhile to be attempted in future clinical studies.
Keywords adrenocortical carcinoma · target-specific therapy . EGFR . Ras/ERK pathway . PI3K/Akt pathway . immunohistochemistry
Introduction
Adrenocortical carcinoma (ACC) is a rare tumor with a reported annual incidence of 0.5 to 2 cases per million [1].
The great majority of the patients with ACC present symptoms related to hormonal activity. Complete surgical excision is still considered the most effective therapy for ACC [1]. However, gradual clinical onset and/or development of these endocrine-related symptoms may be unrecognized for a long time and many of the patients present with advanced clinical stages including distant metastasis. Chemotherapy in combination with mitotane, an anti-adrenocortical agent, has been frequently utilized with varying response rates in these patients [2]. It is, however, true that clinical outcome or prognosis in these cases is still markedly poor or dismal despite development of these aggressive therapies above.
The detailed study of molecular pathways related to tumor growth, metastasis, and infiltration resulted in the develop- ment of target-specific therapy. This therapy inhibits or suppresses the proliferation of tumor cells by interfering with specific molecules expressed in the tumor cells, rather than by nonspecifically interfering with rapidly proliferating cells as in conventional chemotherapy. Target-specific therapy has been reported to be more effective than current chemotherapy and less harmful to normal cells in several cancers [3, 4]. However, the presence of these specific targets is prerequi- site or mandatory for these agents to exert any therapeutic effects and it is necessary to evaluate the presence or absence of these in tumor tissues, especially in archival tissue materials in cases of recurrent or metastatic cases. The most studied signaling targets in the context of current drug development which may provide candidates of potential therapeutic agents include erbB family, vascular endothelial growth factors (VEGFs) and its receptors, and cytoplasmic kinases lying on Ras/extracellular signal-regulated kinases (ERK) and phosphatidylinositol-3 kinase (PI3K)/Akt path- way [5, 6]. Therefore, in this study, we immunolocalized ten specific molecules related to the Ras/ERK and PI3K/Akt pathway in 41 ACC cases, 54 adrenocortical adenoma (ACA) cases, and five nonpathological adrenal glands and determined which markers were more abundant in ACC than ACA or normal adrenals as a first step toward exploring the possibility of target-specific therapy in the patients with ACC. These markers include vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2, epidermal growth factor receptor (EGFR), human EGFR-related 2, extracellular signal-regulated kinases 1/2, Akt, mammalian target of rapamycin (mTOR), p70S6 kinase, S6 ribosomal protein, and 4E binding protein.
Materials and Methods
Adrenals
The total of 95 cases of adrenocortical neoplasms (41 ACC cases, 54 ACA cases) and five nonpathological adrenal
glands were retrieved from surgical pathology and autopsy files including consultation materials between 1976 and 2007 from Department of Pathology, Tohoku University Hospital (Sendai, Japan). Clinicopathological features of the cases examined are summarized in Table 1. The data on survival and mutation or copy number are not shown because the information about consultation cases is restrict- ed. All of the specimens had been fixed in 10% formalin at room temperature and embedded in paraffin wax. The diagnosis of ACC was made according to Weiss’ criteria of adrenocortical malignancy [7]. In brief, the criteria consist of histological scoring systems evaluating multiple histo- logical parameters obtained from evaluation in hematoxy- lin-eosin stained histological slides. A tumor is defined as ACC when three or more of the following criteria are met: (1) nuclear grade III or IV, (2) mitotic rate six or more per 50 high power fields, (3) atypical mitosis, (4) clear cells less than 25%, (5) a diffuse architecture pattern in greater than one third of the tumor, (6) confluent necrosis, (7) venous invasion, (8) sinusoidal invasion, and (9) capsular invasion. The criteria are relatively straightforward and considered the most effective standard for diagnosis of adrenocortical malignancy [8]. Research protocols for this retrospective study were approved by the Ethics Committee of Tohoku University School of Medicine (2007-360).
Surrogate Markers
The erbB family and VEGF receptors (VEGFRs) or receptor tyrosine kinases are cell surface transmembrane proteins, which activate intracellular signaling pathways with or without bindings of ligands, resulting in induction or stimulation of cell proliferation, migration, invasion, and other biological activities [9, 10]. The erbB family receptors include epidermal growth factor receptor (erbB1), human EGFR-related 2 (HER2; erbB2), HER3 (erbB3), and HER4 (erbB4) [11, 12]. VEGF family, a putative target of bevacizumab, includes six secreted glycoproteins referred to as VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placenta growth factors 1 and 2. VEGFA is a mitogen and survival factor for vascular endothelial cells [13],which interacts with two receptor tyrosine kinases including VEGFR1 and VEGFR2 [10]. VEGFR2 is the major mediator of the effects of VEGFA [13].
ERK1/2 and Akt represent potential biomarkers to assess the clinical activity of lapatinib in human breast adenocar- cinoma cell line, BT474 [14-16]. ERK1/2, also known as p42/p44 mitogen-activated protein kinases (p42/p44MAPK), is one of the factors related to the cell proliferation and differentiation [17]. Akt, also referred to as protein kinase B, plays an important role in controlling the balance between survival and apoptosis of the cells [18]. Increased phosphor- ylation of both Akt and p70S6 kinase (p70S6k) was
| ACC | ACA | Nonpathological adrenal gland | |
|---|---|---|---|
| Case | 41 | 54 | 5 |
| Age | 0 years 7 months-69 years | 21-83 years | 0 years 6 months-77 years |
| Median | 37 years | 47 years | 63 years |
| Gender | |||
| Male | 12 (29%) | 20 (37%) | 5 (100%) |
| Female | 29 (71%) | 34 (63%) | 0 (0%) |
| Cushing syndrome | 23 | 14 | |
| Pre-Cushing syndrome | 0 | 14 | |
| Aldosteronism | 5 | 25 | |
| Virilization | 11 | 0 | |
| Nonfunctioning | 5 | 1 | |
| DOC producing | 2 | 0 |
DOC deoxycorticosterone
especially reported in Kaposi’s sarcoma, which was most likely the result of the activation of VEGF receptors [15]. mTOR is a sensor for adenosine triphosphate and amino acids and is involved in cellular growth and homeostasis [19]. p70S6k is a direct target of mTOR and plays a role in tumor invasiveness, motility, and angiogenesis. The effect of p70S6k on mRNA translation is indirect via intermediates such as S6RP [20]. In addition, 4E binding protein (4EBP) is the other direct target of mTOR [20]. 4EBP normally binds eukaryotic translation initiation factor 4E. Phosphorylation of 4EBP by mTOR resulted in the activation of cap- dependent translation.
These ten molecules above are therefore considered to represent potential surrogate markers for target-specific therapy at least in advanced ACC cases at this juncture. Therefore, in
this study, we immunolocalized ten specific molecules related to the Ras/ERK and PI3K/Akt pathway. No clinical trials with agents targeted these molecules have been reported in the literature at this juncture to the best of our knowledge.
Antibodies for Immunohistochemistry
The properties of antibodies employed in immunohistochemis- try were summarized in Table 2. Antibodies for the cytoplasmic molecules detect only activated forms of the protein.
Immunohistochemistry
Immunohistochemical procedures were performed employing polymer signal amplification system with EGFR pharmDx kit
| Product name | Company | Catalog number | IgG typing | Dilution | Positive control | |
|---|---|---|---|---|---|---|
| VEGF (A-20) | Santa Cruz Biotechnology, Inc. | (Santa Cruz, CA, USA) | sc-152 | Polyclonal rabbit | 1/500 | Breast carcinoma |
| VEGF Receptor 2 | Abcam Ltd. | (Cambridge, UK) | ab2349 | Polyclonal rabbit | 1/100 | Angiosarcoma |
| EGFR pharmDx Kit | DakoCytomation | (Glostrup, Denmark) | K1492 | Monoclonal mouse | 1/1 | Control cell slide |
| c-erbB-2 | Invitrogen Corporation | (Carlsbad, CA, USA) | 08-1203 | Monoclonal mouse | 1/1 | Breast carcinoma |
| Phospho-p44/42 MAPK (Thr202/Tyr204) | Cell Signaling Technology, Inc. | (Danvers, MA, USA) | #4376 | Monoclonal rabbit | 1/100 | Colon carcinoma |
| Phospho-Akt (Ser473) | Cell Signaling Technology, Inc. | (Danvers, MA, USA) | #4051 | Monoclonal mouse | 1/200 | Lung carcinoma |
| Phospho-mTOR (Ser2448) | Cell Signaling Technology, Inc. | (Danvers, MA, USA) | #2971 | Polyclonal rabbit | 1/50 | Lung SCC |
| Phoapho-p70 S6 Kinase (Thr389) | Cell Signaling Technology, Inc. | (Danvers, MA, USA) | #9206 | Monoclonal mouse | 1/1,000 | Breast carcinoma |
| Phospho-S6 Ribosomal Protein (Ser240/244) | Cell Signaling Technology, Inc. | (Danvers, MA, USA) | #2215 | Polyclonal rabbit | 1/100 | Breast carcinoma |
| Phospho-4E-BP1(Thr70) | Cell Signaling Technology, Inc. | (Danvers, MA, USA) | #9455 | Polyclonal rabbit | 1/50 | SCCHN |
SCC squamous cell carcinoma, SCCHN squamous cell carcinoma of head and neck
(DakoCytomation, Glostrup, Denmark) for EGFR, the streptavidin-biotin amplification method using an EnVision + kit (DakoCytomation) for mTOR, and a Histofine kit (Nichirei, Tokyo, Japan) for the others. Three-micron slices obtained from paraffin-embedded specimens were deparaffi- nized. Antigen retrieval was performed by heating the slides placed on glue coated glasses in a microwave at 500 W for 15 min in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0) for VEGFR2, ERK 1/2, Akt, mTOR, p70S6k, S6RP, and 4EBP and then allowed to cool down for 1 h at room temperature. Dilutions of the primary antibodies used in this study were determined based on the results of previously reported studies as well as the manu- factures’ recommendations (Table 2) [21-28]. The antigen- antibody complexes were visualized with 3, 3’-diamino- benzidine solution [1 mM 3, 3’-diaminobenzidine, 50 mM Tris-HCl buffer (pH 7.6)] and counterstained with hema- toxylin. Normal rabbit or mouse immunoglobulin G was also used in place of the primary antibodies as a negative control. Tissues used as a positive control were summa- rized in Table 2.
The cells were considered positive, when immunoreac- tivity was detected in the cytoplasm for VEGFA, ERK 1/2, Akt, mTOR, p70S6k, S6RP, and 4EBP and on cell membrane for VEGFR2, EGFR, and HER2. Immunoreac- tivity was evaluated by semiquantitative grading according to the percentage of immunopositive cells, and all of specimens were tentatively categorized into six groups (0,
0%; 1, 1% to 5%; 2, 6% to 25%; 3, 26% to 50%; 4, 51% to 75%; 5, 76% to 100%). The findings were evaluated by two of the authors (M.N. and H.S.) and both inter- and intraobserver differences were less than 5%.
Statistical Analysis
The data obtained in this study were assessed by Z test for the statistical comparison between ACC and ACA cases. The possible correlations between the status of immunoreactivity and ages at diagnosis or scores of Weiss’ criteria of adrenocortical malignancy in ACC were examined by Spear- man’s rank correlation. The possible correlations between the status of immunoreactivities and other clinicopathological features in ACC were examined using ×2 test. All statistical analyses in this study were performed using StatMate III (ATMS, Tokyo, Japan). Statistical significance was deter- mined as less than 0.05 level of probability (P<0.05).
Results
Results were summarized in both Fig. 1 and Table 3.
Immunohistochemistry in Nonpathological Adrenal Glands
VEGFA, VEGFR2, ERK1/2, Akt, mTOR, and S6RP immu- noreactivities were abundant in adrenocortical parenchymal
(a)
(b)
20um
20um
(c)
-20um
| ACC | ACA | Nonpathological adrenal gland | |
|---|---|---|---|
| VEGFA | 2.05±1.22 | 2.15±1.51 | 5.00±0.00 |
| VEGFR2 | 0.39±0.97 | 0.57±0.90 | 3.40±2.19 |
| EGFR | 2.44±1.98 | 1.43±1.22 | 0.30±1.30 |
| HER2 | 0 | 0 | 0 |
| ERK1/2 | 0.15±0.36 | 0.26±0.52 | 2.80±1.64 |
| Akt | 0.56±0.92 | 0.52±1.02 | 4.20±1.30 |
| mTOR | 0.41±1.00 | 0.74±1.10 | 4.60±0.55 |
| p70S6k | 0.15±0.42 | 0.28±0.45 | 1.80±1.30 |
| S6RP | 0.34±0.69 | 0.46±0.66 | 4.60±0.89 |
| 4EBP | 0.22±0.57 | 0.28±0.49 | 1.20±1.64 |
cells. EGFR, p70S6k, and 4EBP immunoreactivities were sporadically detected in adrenocortical parenchymal cells. HER2 immunoreactivity was not detected at all in all the cases examined. Only ERK1/2 expression was detected in adrenal
medulla in one case and all other immunoreactivities examined in this study were negative in adrenal medullar.
Immunohistochemistry in ACC and ACA
EGFR immunoreactivity was significantly more abundant in ACC than ACA or nonpathological adrenal gland (P<0.01; Fig. 1a-c). There were no significant differences in the status of immunoreactivities of other molecules examined between ACC and ACA.
In the cases of ACC, the correlation between the status of immunoreactivity of these molecules and age at diagnosis, gender, hormonal features, or score of Weiss’ criteria were examined, respectively. VEGFA immunoreac- tivity was significantly more abundant in the cases with higher score of Weiss’ criteria (P<0.01; Fig. 2d, e) and in the cases with positive findings in mitotic rate, atypical mitosis, or venous invasion, respectively (P<0.05, P<0.05,
(d)
(e)
— 20um
20μm
(f)
8
(g)
20um
20um
(h)
- 20pm
P<0.01). No significant correlations were detected between the status of other molecules and the features above.
Discussion
In this study, we immunolocalized the specific molecules located on the Ras/ERK and PI3K/Akt pathway in adrenocortical neoplasms. The status of EGFR immunore- activity was significantly more abundant in ACC than ACA or nonpathological adrenal gland. EGFR expression was also reported to be markedly elevated in ACC [29], which is consistent with results of our present study. Cetuximab, gefitinib, and erlotinib, agents targeting on EGFR, are clinically available and have been reported to be adminis- tered in the patients with cancer, like nonsmall cell lung cancer [4]. The relative abundance of EGFR in ACC suggests that these agents may provide clinical benefits. However, recent studies also demonstrated that, in contrast to the status of HER2 expression in the tumor cells, EGFR expression itself is by no means a robust predictor of clinical response to target therapies against EGFR, because some patients with EGFR overexpressing tumors have been reported to be refractory to EGFR inhibitors, while on the other hand, patients with a low degree of EGFR expression still respond to EGFR antagonists [5]. In addition, EGFR gene mutations have been reported in the patients with nonsmall cell lung cancer and the status of these mutations may be correlated with the clinical responses to the tyrosine kinase inhibitors such as gefitinib [30]. In addition, recent results of CRYSTAL phase III trial also demonstrated that k-ras wild-type status increased progression-free survival and response rate in patients with metastatic colorectal cancer receiving cetuximab therapy or anti-EGFR antibody. It is also interesting to note that results of previous studies of k-ras mutations demonstrated the absence of mutations in the case of ACC [31]. Lin et al., however, reported that mutations of the k-ras gene were detected in adrenocortical tumor but their study did not include any patients of ACC [32]. These findings all suggest the potential response to the treatment with cetuximab in the patients with ACC. It is true that further investigations are required to apply these tyrosine kinase inhibitors for the therapy toward the ACC patients associated with EGFR abnormalities in the carci- noma cells including point mutations or other genetic abnormalities of EGFR itself. However, at least, results of our present study suggest that anti-EGFR therapies for the patients with adrenocortical carcinoma are considered worthwhile to pursue further investigations.
The criteria of Weiss are a universally well-established or validated histopathological scoring system to make a definitive final diagnosis of ACC in surgically resected specimens of adrenocortical tumors. VEGFA expression in
the tumor cells was positively correlated with scores of Weiss’ criteria. The patients with ACC were reported to be associated with markedly high circulating VEGF levels, significantly higher than the patients with other adrenocor- tical disorders [33]. In our present study, the status of VEGFA immunoreactivity was positively correlated with some score of Weiss’ criteria. In particular, the status of VEGFA immunoreactivity in carcinoma cells was signifi- cantly higher in the cases associated with venous invasion, high mitotic rate, or atypical mitosis. Weiss reported that among the nine histological factors in his criteria, mitotic activity, especially with atypical forms, and venous inva- sion were correlated best with metastasizing or recurring behavior of the resected adrenocortical tumors [7]. There- fore, the presence of VEGFA in carcinoma cells is considered to be associated with aggressive biological behavior in the patients with ACC. However, the difference of VEGFA immunoreactivity between ACC and ACA was not necessarily statistically significant, and its relatively abundant expression was also detected in nonpathological adrenal cortical parenchymal cells and it awaits further investigations for clarification.
There were no significant differences in the status of immunoreactivities between ACC and ACA or among the different histopathological parameters in ACC in other molecules studied. Abundant immunoreactivity for HER2, ERK1/2, Akt, mTOR, and p70S6k has been reported in several types of cancer cell lines or tissues [34, 35]. The status of the immunoreactivity for HER2 and ERK1/2 was correlated with the presence of mutations in some malig- nancies [36, 37]. Therefore, the analysis of abnormalities of these molecules at the DNA levels may provide further information regarding the possibility of potential targets in the patients with ACC.
Acknowledgments We appreciate Mr. Katsuhiko Ono, Ms. Toshie Suzuki, Ms. Miki Mori (Department of Pathology, Tohoku University School of Medicine), and Ms. Yuko Sagara (Department of Pediatric Surgery, Tohoku University School of Medicine) for skillful technical assistance.
Conflict of Interest The authors indicated no potential conflict of interest and no financial interests relating to the material in the manuscript.
References
1. Rescorla FJ. Malignant adrenal tumors. Semin Pediatr Surg 15:48-56, 2006. doi:10.1053/j.sempedsurg.2005.11.008.
2. Terzolo M, Angeli A, Fassnacht M, et al. Adjuvant mitotane treatment for adrenocortical carcinoma. N Engl J Med 356:2372- 80, 2007. doi:10.1056/NEJMoa063360.
3. Viani GA, Afonso SL, Stefano EJ, De Fendi LI, Soares FV. Adjuvant trastuzumab in the treatment of her-2-positive early
breast cancer: a meta-analysis of published randomized trials. BMC Cancer 7:153-63, 2007. doi:10.1186/1471-2407-7-153.
4. Thatcher N. The place of targeted therapy in the patient management of non-small cell lung cancer. Lung Cancer 57 (Suppl 2):S18-23.11, 2007.
5. Bianco R, Melisi D, Ciardiello F, Tortora G. Key cancer cell signal transduction pathways as therapeutic targets. Eur J Cancer 42:290-4, 2006. doi:10.1016/j.ejca.2005.07.034.
6. Press MF, Lenz HJ. EGFR, HER2 and VEGF pathways: validated targets for cancer treatment. Drugs 64:2045-75, 2007.
7. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 8:163- 9, 1984. doi:10.1097/00000478-198403000-00001.
8. Sasano H, Suzuki T, Moriya T. Recent advances in histopathology and immunohistochemistry of adrenocortical carcinoma. Endocr Pathol 17:345-54, 2006. doi:10.1007/s12022-006-0006-0.
9. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 19:3159-67, 2000. doi:10.1093/emboj/19.13.3159.
10. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 9:669-76, 2003. doi:10.1038/nm0603-669.
11. Sebastian S, Settleman J, Reshkin SJ, Azzariti A, Bellizzi A, Paradiso A. The complexity of targeting EGFR signaling in cancer: from expression to turnover. Biochem Biophys Acta 1766:120-39, 2006.
12. Moasser MM. Targeting the function of the HER2 oncogene in human cancer therapeutics. Oncogene 26:6577-92, 2007. doi:10.1038/sj.onc.1210478.
13. Roskoski R Jr. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol 62:179-213, 2007. doi:10.1016/j.critrevonc.2007.01.006.
14. Xia W, Mullin RJ, Keith BR, et al. Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activa- tion of EGFR/erbB2 and downstream Erk1/2 and AKT pathways. Oncogene 21:6255-63, 2002. doi:10.1038/sj.onc.1205794.
15. Stallone G, Schena A, Infante B, et al. Sirolimus for Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 352:1317- 23, 2005. doi:10.1056/NEJMoa042831.
16. Duran I, Kortmansky J, Singh D, et al. A phase II clinical and pharmacodynamic study of temsirolimus in advanced neuroendo- crine carcinomas. Br J Cancer 95:1148-54, 2006. doi:10.1038/sj. bjc.6603419.
17. Pearson G, Robinson F, Beers GT, et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22:153-83, 2001. doi:10.1210/er.22.2.153.
18. Brazil DP, Yang ZZ, Hemmings BA. Advances in protein kinase B signaling: AKTion on multiple fronts. Trends Biochem Sci 29:233-42, 2004. doi:10.1016/j.tibs.2004.03.006.
19. Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRAP/mTOR. Genes Dev 15:807-26, 2001. doi:10.1101/ gad.887201.
20. Mamane Y, Petroulakis E, LeBacquer O, Sonenberg N. mTOR, translation initiation and cancer. Oncogene 25:6416-22, 2006. doi:10.1038/sj.onc.1209888.
21. Kaio E, Tanaka S, Kitadai Y, et al. Clinical significance of angiogenic factor expression at the deepest invasive site of advanced colorectal carcinoma. Oncology 64:61-73, 2003. doi:10.1159/000066511.
22. Derecskei K, Moldvay J, Bogos K, Tímár J. Protocol modifica- tions influence the result of EGF receptor immunodetection by EGFR pharmDx in paraffin-embedded cancer tissues. Pathol Oncol Res 12:243-6, 2006.
23. Yamashita S, Suzuki S, Nomoto T, et al. Linkage and microarray analyses of susceptibility genes in ACI/Seg rats: a model for prostate cancers in the aged. Cancer Res 65:2610-6, 2005. doi:10.1158/0008-5472.CAN-04-2932.
24. Malik SN, Brattain M, Ghosh PM, et al. Immunohistochemical demonstration of phosphor-Akt in high Gleason grade prostate cancer. Clin Cancer Res 8:1168-71, 2002.
25. Robb VA, Karbowniczek M, Klein-Szanto AJ, Henske EP. Activation of the mTOR signaling pathway in renal clear cell carcinoma. J Urol 177:346-52, 2007. doi:10.1016/j.juro.2006. 08.076.
26. Cen L, Hsieh FC, Lin HJ, Chen CS, Qualman SJ, Lin J. PDK-1/ AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound. Br J Cancer 97:785-91, 2007. doi:10.1038/sj.bjc.6603952.
27. Miyata H, Chiang AC, Vinters HV. Insulin signaling pathways in cortical dysplasia and TSC-tubers: tissue microarray analysis. Ann Neurol 56:510-9, 2004. doi:10.1002/ana.20234.
28. Li X, Alafuzoff I, Soininen H, Winblad B, Pei JJ. Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer’s disease brain. FEBS J 272:4211-20, 2005. doi:10.1111/j.1742-4658.2005.04833.x.
29. Sasano H, Suzuki T, Shizawa S, Kato K, Nagura H. Transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor expression in normal and diseased human adrenal cortex by immunohistochemistry and in situ hybridization. Mod Pathol 7:741-6, 1994.
30. Loprevite M, Tiseo M, Chiaramondia M, et al. Buccal mucosa cells as in vivo model to evaluate gefitinib activity in patients with advanced non small cell lung cancer. Clin Cancer Res 13:6518- 26, 2007. doi:10.1158/1078-0432.CCR-07-0805.
31. Yashiro T, Hara H, Fulton NC, Obara T, Kaplan EL. Point mutations of ras genes in human adrenal cortical tumors: absence in adrenocortical hyperplasia. World J Surg 18:455-61, 1994. doi:10.1007/BF00353735.
32. Lin SR, Tsai JH, Yang YC, Lee SC. Mutation of K-ras oncogene in human adrenal tumours in Taiwan. Br J Cancer 77:1060-5, 1998.
33. Zacharieva S, Atanassova I, Orbetzova M, et al. Circulating vascular endothelial growth factor and active rennin concentra- tions and prostaglandin E2 urinary excretion in patients with adrenal tumors. Eur J Endocrinol 150:345-9, 2004. doi:10.1530/ eje.0.1500345.
34. Lin F, Zhang PL, Yang XJ, Prichard JW, Lun M, Brown RE. Morphoproreomic and molecular concomitants of an overex- pressed and activated mTOR pathway in renal cell carcinomas. Ann Clin Lab Sci 36:283-93, 2006.
35. Molinolo AA, Heiwitt SM, Amornphimoltham P, et al. Dissecting the Akt/mammalian target of rapamycin signaling network: emerging results from the head and neck cancer tissue array initiative. Clin Cancer Res 13:4964-73, 2007. doi:10.1158/1078- 0432.CCR-07-1041.
36. Pérez-Tenorio G, Alkhori L, Olsson B, et al. PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer. Clin Cancer Res 13:3577- 84, 2007. doi:10.1158/1078-0432.CCR-06-1609.
37. Schmitz KJ, Wohlschlaeger J, Alakus H, et al. Activation of extracellular regulated kinases (ERK1/2) but not AKT predicts poor prognosis in colorectal carcinoma and is associated with k- ras mutations. Virchows Arch 450:151-9, 2007. doi:10.1007/ s00428-006-0342-y.