Differential Expression of Epidermal Growth Factor Receptor, C-Met, and HER2/Neu in Chordoma Compared with 17 Other Malignancies

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ORIGINAL ARTICLE Differential Expression of Epidermal Growth Factor Receptor, c-Met, and HER2/neu in Chordoma Compared With 17 Other Malignancies

Paul M. Weinberger, MD; Ziwei Yu, MD; Diane Kowalski, MD; John Joe, MD; Phillip Manger, MD; Amanda Psyrri, MD; Clarence T. Sasaki, MD

Objective: To examine the expression of c-Met, c- Results: Most chordomas displayed strong expression Erb-b2 (HER2/neu), and epidermal growth factor (EGFR) of EGFR and c-Met, whereas a variable level of expres- in a cohort of 12 chordomas, based on the current and sion of HER2/neu was seen. In addition, we noted a strong future availability of targeted molecular inhibitors. correlation between EGFR and c-Met expression, especially for primary chordomas (P=.006). Design: Protein expression levels were analyzed by immunohistochemical analysis from archival tissue using multitumored tissue microarray and by Spearman rank Conclusions: Chordomas, like many other solid-tissue correlation test. tumors, express HER2/neu, EGFR, and the hepatocyte growth factor/scatter factor receptor c-Met. Most chor- Setting: Tertiary care facility. domas had strong expression of both the hepatocyte growth factor/scatter factor receptor and EGFR. Inhibi- Subjects: We assembled the cohort of 12 chordomas from tors to EGFR are already in clinical use for other solid- patients treated at the Yale–New Haven Hospital from 1986 to 2003 and compared them with 51 other tumors tissue tumors and represent a potentially viable experi- from 17 assorted solid tissue tumor types along with 1 mental treatment option for refractory chordoma. Further sample of healthy tissue from each site. studies are required to investigate these findings.

Main Outcome Measure: The expression of c-Met, HER2/neu, and EGFR. Arch Otolaryngol Head Neck Surg. 2005;131:707-711

HORDOMAS ARE RARE NEO- sive surgical excision followed by local ir- plasms arising from no- radiation, but even with the best treat- tochordal remnants in the ments available, overall survival time midline skeletal axis. The remains roughly 5 years.1 most common sites are the Given the rare nature of these tumors, skullC base and the sacrococcygeal region.1 current understanding of molecular Because there is an annual incidence of just marker expression in chordomas is lim- 1 in 2 million for skull base chordomas, ited. With the recent introduction of tar- relatively little is known about the molecu- geted molecular therapeutics into clini- lar biology of these tumors.2 They are typi- cal practice, an enhanced understanding cally slow-growing tumors, and initial of the molecular biology of chordomas is Author Affiliations: Section of symptoms are usually related to local pro- needed. One important cell-signaling path- Otolaryngology gression of the disease with subsequent way in cancer progression involves a class (Drs Weinberger, Yu, Joe, compression of adjacent structures. Vague of molecules known as receptor tyrosine Manger, and Sasaki), headache, visual changes, and cranial nerve kinases (RTKs). These molecules serve as Departments of Pathology palsies are common manifestations of skull membrane-bound cytokine receptors and (Dr Kowalski) and Medical base chordomas. By the time most pa- initiate an intracellular signaling cascade Oncology (Dr Psyrri), Yale tients report these symptoms, most chor- on ligand binding. There are multiple ex- University, New Haven, Conn; 1 and Department of domas have already grown quite large. amples of RTK proto-oncogenes, includ- Otolaryngology, Medical The natural course of chordoma is quite ing c-sis (the platelet-derived growth fac- College of Georgia, Augusta grim; most patients do not survive 10 years tor receptor), the erb-b/HER epidermal (Dr Weinberger). because of high local recurrence rates.3,4 growth factor receptor family (EGFR), and Financial Disclosure: None. The current treatment of choice is aggres- c-met (the hepatocyte growth factor/

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Target Identifier Type Manufacturer Dilution Source c-Met c28 Rabbit polyclonal Santa Cruz Biotechnology, Santa Cruz, Calif 1:1000 Paciucci et al,11 1998 c-Erb-b2 (HER2/neu) a0485 Rabbit polyclonal Dako Cytomation, Carpinteria, Calif 1:200 Pai-Scherf et al,12 1999 EGFR 2232 Rabbit polyclonal Cell Signaling Technologies, Beverly, Mass 1:100 Endo et al,13 2002

Abbreviation: EGFR, epidermal growth factor.

scatter factor receptor).5,6 All of these RTKs have been be cored. The cores were placed on the recipient microarray demonstrated to possess mitogenic and possible trans- block using a tissue microarrayer (Beecher Instruments, Sil- forming qualities on activation and signal transduction ver Spring, Md). The tissue microarray was then cut to yield via tyrosine kinase phosphorylation.7,8 Inhibitors to mem- 5-µm sections, which were placed on glass slides using an ad- bers of the erb-b/HER family EGFR and HER2/neu are hesive tape transfer system (Instrumedics, Inc, Hackensack, NJ) with UV cross-linking. already in clinical use and have demonstrated efficacy in other more common solid-tissue tumors, including breast and lung cancer.9 In addition, use of inhibitors of c-met IMMUNOHISTOCHEMICAL ANALYSIS is currently planned for clinical trials and in the future may be approved for widespread clinical use. Because of All slides were processed simultaneously in identical conditions using standard methods. Chordoma slides were pro- the rare nature of chordomas, large-scale clinical trials cessed as standard 5-µm tissue sections mounted on sialinized of these agents in chordomas seem unlikely. If a molecu- glass slides (Dako, Carpenteria, Calif). Multitumor tissue mi- lar basis for predicted efficacy of a targeted agent could croarray slides were prepared as described in the previous sub- be discovered, however, such agents may represent a vi- section. Slides were deparaffinized in xylene followed by 2 rinses able experimental treatment option for individual pa- in absolute ethanol. Following a 1-minute rinse in deionized tients with refractory disease. water, heat-induced epitope retrieval was performed by pres- Basing our study on the current or future availability sure cooking in citrate buffer, pH 6.0, for 7 minutes. Nonspe- of targeted molecular inhibitors, we examined the ex- cific primary antibody interactions were blocked by incuba- pression of c-Met, c-Erb-b2 (HER2/neu), and epidermal tion with 0.3% bovine serum albumin in 0.1M tris-buffered saline growth factor receptor (EGFR) in a cohort of 12 chor- (BSA/TBS), pH 8.0, for 30 minutes at room temperature. Slides were then incubated with primary antibodies diluted in BSA/ domas and compared them with 51 tumors from 17 as- TBS overnight at 4°C (Table 1). Slides were then washed 3 sorted tumor types. Given the limited number of chor- times (for 5 minutes each time) with BSA/TBS containing 0.05% domas available for analysis, these other tumor types Tween-20 to remove unbound primary antibody. Goat anti- provided an internal comparison for the results. rabbit secondary antibody conjugated to a horseradish peroxi- dase decorated dextran-polymer backbone (Envision; Dako) was METHODS then applied for 1 hour at room temperature. Diaminiobenzi- dine chromagen was used to visualize antibody binding. All slides were run simultaneously under identical conditions and in- CHORDOMA COHORT SELECTION cluded negative control (no primary antibody) slides. Following institutional review board approval, we assembled the cohort of chordoma tumors from all patients with a patho- IMMUNOHISTOCHEMICAL SCORING logical diagnosis of chordoma treated at Yale–New Haven Hos- FOR EGFR, c-Erb-b2, AND c-Met pital, New Haven, Conn, from 1986 to 2003. All patients were included; we had no exclusion criteria other than lack of avail- Consensus scoring by 2 observers (P.M.W. and D.K.) deter- able material for immunohistochemical analysis. mined molecular marker expression. Membranous staining in- tensity was evaluated and scored on an ordinal 0 to 3ϩ scale MULTITUMOR TISSUE MICROARRAY for c-Met, EGFR, and HER2/neu. Occasional cytoplasmic stain- CONSTRUCTION ing was seen with c-Met but was not included in scoring. We scored each paraffin section of tumor and each tumor core using a 4-tiered grading system: 0, no staining; 1ϩ, weak stain- A multitumor tissue microarray had been previously as- ing with incomplete circumferential membrane staining; 2ϩ, sembled and was used for comparison of chordoma molecular moderate staining with complete circumferential membrane marker expression with various other malignancies. We as- staining; and 3ϩ, strong staining with complete circumferen- sembled the multitumor cohort from patients treated at Yale– tial membrane staining. Samples with scores of 2ϩ or 3ϩ were New Haven Hospital between 1980 and 1999 with the appro- considered overexpression of that protein. priate pathological diagnosis. We included 3 tumors of each type plus 1 normal tissue for each site. Following institutional review board approval, we constructed the tissue microarray STATISTICAL ANALYSIS as previously described10 and included 51 tumor cases from varying sites. We obtained tissue cores from paraffin-embedded for- We evaluated correlation of marker expression by Spearman malin-fixed tissue blocks from the archives at the Department rank correlation test. All calculations and analyses were per- of Pathology, Yale–New Haven Hospital. formed with SPSS 11.5 for Windows (SPSS Inc, Chicago, Ill) A pathologist (D.K.) reviewed slides from all blocks to se- and, where appropriate, were 2-tailed. Significance was con- lect representative areas of invasive tumor or normal tissue to sidered to be PϽ.05.

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Molecular Marker Expression

Patient No./Sex/Age, y Tumor Location c-Met Erb-b2 EGFR 1/F/56 Sacrum 3 0 3 2/F/50 Other vertebral 1 3 1 3/M/60 Other vertebral 2 1 1 3/Recurrence 3 0 2 4/M/78 Skull base 3 3 3 5/F/68 Sacrum 3 0 3 6/F/49 Sacrum 3 0 2 7/F/68 Sacrum 3 2 2 8/F/49 Skull base 3 3 3 8/Recurrence 3 0 3 9/M/16 Skull base 2 2 2 10/F/86 Sacrum 3 3 3

Abbreviation: EGFR, epidermal growth factor.

RESULTS A B

COHORT DEMOGRAPHICS

After review of the Department of Pathology records at Yale–New Haven Hospital, we identified 13 surgical specimens from 11 patients diagnosed as having chordomas. One of these blocks was unavailable, leaving 12 chordoma specimens (from 10 patients). Two patients expe- rienced recurrence and subsequent resection. These recurrent specimens were included in the cohort but separated out for statistical analysis, leaving 10 unique C D specimens for comparison. The patients (7 women and 3 men) ranged in age from 16 to 86 years (median age, 58 years). Locations of the chordomas were skull base (3), sacrum (5), and other vertebra (2). Demographic in- formation is summarized in Table 2. The multitumor tissue microarray consisted of 51 tumors of 17 malignancies (3 tumors from each type) along with 1 healthy tissue from each site. Tumor types included head and neck squamous cell, renal cell, and gas- tric carcinomas; melanoma; prostate, brain, ovarian, germ Figure 1. Receptor tyrosine kinase (RTK) expression. With immunoperoxidase staining, we determined expression for RTKs using cell, colon, bladder, endometrial, thyroid, hepatic, pan- primary antibody to c-Met, epidermal growth factor (EGFR), or HER2/neu. creatic, and lung cancers; lymphoma; and sarcoma. A, Chordoma with moderate (1ϩ) HER2/neu expressing chordoma. B, Chordoma with moderate (2ϩ) c-Met expressing chordoma. C, Chordoma with strong (3ϩ) EGFR expressing chordoma. D, Moderate (2ϩ) EGFR IMMUNOHISTOCHEMICAL ANALYSIS FOR expressing ovary for comparison. HER2/neu, c-Met, AND EGFR EXPRESSION

Twelve (100%) of 12 chordoma tumors and 37 (73%) A representative micrograph of moderate (2ϩ) HER2/ of 51 comparison tumor specimens had sufficient rep- neu expression in chordoma is shown in Figure 1A. resentative tissue for analysis of HER2/neu expression. For c-Met, 12 (100%) of 12 chordomas and 39 (76%) Chordomas displayed a spectrum of intensities ranging of 51 comparison tumors had sufficient representative from no (0) expression to very strong (3ϩ) expression tissue for analysis. Chordomas displayed predomi- of HER2/neu. Among primary chordomas there were 3 nately strong expression of c-Met, with no tumors dis- tumors with no expression (0), 1 with mild (1ϩ), 2 with playing absent expression, 1 with mild (1ϩ), 2 with mod- moderate (2ϩ), and 4 with strong (3ϩ) expression. Both erate (2ϩ), and 7 with strong (3ϩ) expression. The recurrent chordomas lacked expression of HER2/neu. recurrent chordoma tumors both displayed strong (3ϩ) Among comparison tumors, prostate and bladder carci- expression of c-Met. Among comparison tumors, gas- noma displayed predominately strong (3ϩ) expression, tric, prostate, ovarian, and colon carcinomas displayed whereas lymphomas, hepatic carcinomas, and sarcomas predominately strong (3ϩ) expression, whereas thy- displayed predominately weak to no staining (0 to 1ϩ). roid carcinoma and lymphomas displayed predomi-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 ducted to determine expression patterns of other poten- 8 A B C tially useful molecular markers. A recent study by Deniz et al17 found that expression levels of basic fibroblast 6 growth factor, transforming growth factor ␣, and fibronectin were all correlated with local recurrence and ag- 18 4 gressive biological behavior. Kilgore and Prayson found no association between cyclin D1, MIB-1, p53, and BCL-2 No. of Tumors 2 and prognosis. In addition, the receptor tyrosine kinase c-Met was found to be highly expressed in chordomas 19 0 in one study of benign and malignant bone tumors. 0 1 2 3 0 1 2 3 0 1 2 3 In the present study, we decided to focus our inves- c-Met HER2/neu EGFR Expression Expression Expression tigations on the differential expressions of c-Met, EGFR/ HER1, and HER2/neu RTKs between chordomas and a wide variety of tumors. As inhibitors of these RTKs be- Figure 2. Receptor tyrosine kinase expression patterns in primary chordoma (n=10). A, c-Met expression in primary chordoma was noted to be primarily come more widely used in the clinical setting, a better strong (3ϩ). B, HER2/neu expression was variable from no expression to understanding of the signaling pathways in vivo, their strong expression. C, Epidermal growth factor (EGFR) expression was pattern of expression and activation, and their modula- ϩ primarily strong (3 ). tion by these therapies is of critical importance because it may be helpful in predicting the target tumor popula- tion that will benefit from these therapies. nately absent (0) staining. A representative micrograph We found that all 3 of these RTKs are expressed to of moderate (2ϩ) c-Met expression in chordoma is shown varying degrees in chordomas. For EGFR and c-Met, most in Figure 1B. chordomas displayed strong expression, whereas for For EGFR, 12 (100%) of 12 chordomas and 39 (76%) HER2/neu we saw a variety of expression levels. In ad- of 51 comparison tumors had sufficient representative dition, we noted a strong correlation between EGFR and tissue for analysis. Chordomas displayed predomi- c-Met expression, especially for primary chordoma tu- nately strong expression of EGFR, with no tumors dis- mors. playing absent expression, 2 with mild (1ϩ), 3 with mod- The ErbB/HER receptor family is composed of the fol- erate (2ϩ), and 5 with strong (3ϩ) expression. The lowing 4 related receptors: EGFR (Erb-b1/EGFR/ recurrent chordoma tumors displayed moderate (2ϩ) and HER1), Erb-b2 (HER2/neu), Erb-b3 (HER3), and Erb-b4 strong (3ϩ) expression of EGFR. Among comparison tu- (HER4). Ligand binding promotes receptor dimeriza- mors, melanoma, prostate, brain, ovarian, sarcoma, and tion, which results in high-affinity ligand binding, acti- hepatic carcinoma displayed predominately strong (3ϩ) vation of the intrinsic protein kinase activity, and tyro- expression, whereas lymphomas displayed predomi- sine autophosphorylation. These events result in the nately mild (1ϩ) staining. Representative micrographs activation of a signal transduction cascade that is mito- of EGFR expression in chordoma and ovary are shown genic and possibly transforming.20 The c-Met/ in Figure 1C and D. Expression patterns for primary chor- hepatocyte growth factor (HGF) pathway is also very im- domas are summarized in Figure 2. portant in carcinogenesis. The HGF is the ligand of c-Met receptor and is also known as the scatter factor. Signal- CORRELATION OF MARKER EXPRESSION ing pathways activated by c-Met–HGF interaction me- IN CHORDOMA TUMORS diate several cellular processes essential for life. These cellular processes lead to a broad range of biological events, For primary chordoma tumors, there was no relation- including embryological development, wound healing, ship between HER2/neu expression and either c-Met or tissue regeneration, angiogenesis, growth, invasion, and EGFR expression. There was a significant correlation be- morphogenic differentiation.6 Because several of these tween c-Met expression and EGFR expression level physiological processes are known to be crucial in tu- (P=.006, Spearman ␳=0.793). Chordoma tumors that had morigenesis and metastasis, the c-Met/HGF pathway plays high c-Met expression were also likely to have high EGFR a significant role in carcinogenesis. expression. When this analysis was repeated for all tu- Our finding of increased c-Met expression in most mors (primary and recurrent chordomas plus all com- chordomas is important. The chromosomal site of the c- parison tumors), the same relationship was noted. Ex- met proto-oncogene has been located at 7q31.21 Scheil et pression of c-Met and EGFR showed positive correlation al22 found that gains of 7q were among the most com- (P=.03, ␳=0.308), but no relationship was seen be- mon chromosomal alterations noted; 69% of chordo- tween HER2/neu and EGFR or c-Met expression. mas studied demonstrated 7q gain. Thus, gain of c-Met expression via 7q amplification may represent an early COMMENT event in chordoma progression. We also demonstrated a significant and strong corre- Numerous studies14-16 have yielded a consensus on char- lation between c-Met and EGFR expression, especially acteristic immunohistochemical staining parameters that in primary chordomas. Peghini et al23 noted a similar re- define chordomas. These include positive expression of lationship in gastrinomas and found this to correlate with cytokeratin and epithelial membrane antigen. While this aggressive behavior. This is not surprising given the aids pathological diagnosis, few studies have been con- mounting body of evidence that EGFR and c-Met inter-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 act in a variety of ways to modulate downstream mito- 3. Azzarelli A, Quagliuolo V, Cerasoli S, et al. Chordoma: natural history and treat- genic signaling following HGF stimulation.24-26 Schev- ment results in 33 cases. J Surg Oncol. 1988;37:185-191. 25 4. Baratti D, Gronchi A, Pennacchioli E, et al. Chordoma: natural history and re- ing et al demonstrated that specific pharmacologic sults in 28 patients treated at a single institution. Ann Surg Oncol. 2003;10: inhibition of EGFR kinase did not affect c-Met kinase ac- 291-296. tivity but blocked the proliferative effects of HGF bind- 5. de Bono JS, Rowinsky EK. The ErbB receptor family: a therapeutic target for cancer. ing. This is clinically important because it raises the pos- Trends Mol Med. 2002;8(4, suppl):S19-S26. sibility that inhibition of EGFR alone may mediate 6. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003;4:915-925. antitumor effects on multiple RTK signaling pathways. 7. Biscardi JS, Ishizawar RC, Silva CM, Parsons SJ. Tyrosine kinase signalling in Our study’s main limitation was the small sample size, breast cancer: epidermal growth factor receptor and c-Src interactions in breast necessitating a hypothesis-generating experimental de- cancer. Breast Cancer Res. 2000;2:203-210. sign. Despite obvious limitations, such studies are es- 8. de Luca A, Arena N, Sena LM, Medico E. Met overexpression confers HGF- sential in stimulating new research into rare neoplasms. dependent invasive phenotype to human thyroid carcinoma cells in vitro. J Cell Efforts are currently under way to create a larger fol- Physiol. 1999;180:365-371. 9. Averbuch S, Kcenler M, Morris C, Wakeling A. Therapeutic potential of tyrosine low-up experiment through multi-institution collabora- kinase inhibitors in breast cancer. Cancer Invest. 2003;21:782-791. tion. Additional in vitro studies that assess the impact of 10. Rimm DL, Camp RL, Charette LA, Costa J, Olsen DA, Reiss M. Tissue microar- RTK inhibitors on chordoma cell culture would also be ray: a new technology for amplification of tissue resources. Cancer J. 2001; extremely useful. In 2001, Scheil et al22 described the cre- 7:24-31. ation of the only known chordoma cell line, U-CH1. We 11. Paciucci R, Vila MR, Adell T, et al. Activation of the urokinase plasminogen activator/ urokinase plasminogen activator receptor system and redistribution of E- are currently investigating this possibility. cadherin are associated with hepatocyte growth factor-induced motility of pan- Our study demonstrated that EGFR, HER2/neu, and creas tumor cells overexpressing Met. Am J Pathol. 1998;153:201-212. c-Met may play a significant role in the growth of chor- 12. Pai-Scherf LH, Villa J, Pearson D, et al. Hepatotoxicity in cancer patients receiv- domas. Given the natural course of chordomas, any pos- ing erb-38, a recombinant immunotoxin that targets the erbB2 receptor. Clin Can- sible new treatment options deserve exploration. Our re- cer Res. 1999;5:2311-2315. sults may suggest the potential clinical utility of the use 13. Endo A, Nagashima K, Kurose H, Mochizuki S, Matsuda M, Mochizuki N. Sphin- gosine 1-phosphate induces membrane ruffling and increases motility of hu- of RTK inhibitors gefitinib (or erlotinib), trastuzumab, man umbilical vein endothelial cells via vascular endothelial growth factor re- and/or c-Met inhibitors in the treatment of patients with ceptor and CrkII. J Biol Chem. 2002;277:23747-23754. refractory chordomas. These molecule-targeted thera- 14. Bouropoulou V, Bosse A, Roessner A, et al. Immunohistochemical investigation pies will certainly revolutionize cancer therapy. The use of chordomas: histogenetic and differential diagnostic aspects. Curr Top Pathol. of these inhibitors alone or in combination with chemo- 1989;80:183-203. 15. Coffin CM, Swanson PE, Wick MR, Dehner LP. An immunohistochemical com- therapy or radiation shows promise for therapy in a wide parison of chordoma with renal cell carcinoma, colorectal adenocarcinoma, and variety of cancers whose growth depends on aberrant sig- myxopapillary ependymoma: a potential diagnostic dilemma in the diminutive naling via these pathways. Our findings should be viewed biopsy. Mod Pathol. 1993;6:531-538. with caution, but for patients with few other treatment 16. Coindre JM, Rivel J, Trojani M, De Mascarel I, De Mascarel A. Immunohistologi- options, experimental use of one of these agents on an cal study in chordomas. J Pathol. 1986;150:61-63. 17. Deniz ML, Kilic T, Almaata I, Kurtkaya O, Sav A, Pamir MN. Expression of growth “n of 1” trial basis may be a viable option. However, while factors and structural proteins in chordomas: basic fibroblast growth factor, trans- certainly less toxic than traditional chemotherapy, these forming growth factor alpha, and fibronectin are correlated with recurrence. inhibitors are not devoid of adverse effects. A decision Neurosurgery. 2002;51:753-760. to use them experimentally, even for a patient with no 18. Kilgore S, Prayson RA. Apoptotic and proliferative markers in chordomas: a study alternative treatment options, should again be made with of 26 tumors. Ann Diagn Pathol. 2002;6:222-228. 19. Naka T, Iwamoto Y, Shinohara N, Ushijima M, Chuman H, Tsuneyoshi M. Ex- due caution. pression of c-met proto-oncogene product (c-MET) in benign and malignant bone tumors. Mod Pathol. 1997;10:832-838. Submitted for Publication: October 6, 2004; accepted 20. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. March 22, 2005. Oncogene. 2000;19:6550-6565. Correspondence: Clarence T. Sasaki, MD, Department of 21. Glukhova L, Lavialle C, Fauvet D, et al. Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell car- Surgery, Yale University School of Medicine, 333 Cedar cinomas: increased copy number and overexpression of the MET proto-oncogene. St, PO Box 208041, New Haven, CT 06520-8041 (clarence Oncogene. 2000;19:754-761. [email protected]). 22. Scheil S, Bruderlein S, Liehr T, et al. Genome-wide analysis of sixteen chordo- Funding/Support: This study was supported in part by mas by comparative genomic hybridization and cytogenetics of the first human the Doris Duke Charitable Foundation (Dr Wein- chordoma cell line, U-CH1. Genes Chromosomes Cancer. 2001;32:203-211. 23. Peghini PL, Iwamoto M, Raffeld M, et al. 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