fluorescence in situ hybridization gene amplification analysis of egfr and her2 in patients with...

ORIGINAL ARTICLE

FLUORESCENCE IN SITU HYBRIDIZATION GENEAMPLIFICATION ANALYSIS OF EGFR AND HER2IN PATIENTS WITH MALIGNANT SALIVARYGLAND TUMORS TREATED WITH LAPATINIB

Laura Vidal, MD,1 Ming S. Tsao, MD,1 Gregory R. Pond, PhD,1 Ezra E. W. Cohen, MD,2

Roger B. Cohen, MD,1 Eric X. Chen, MD,1 Mark Agulnik, MD,1 Sebastien Hotte, MD,1

Eric Winquist, MD,1 Scott Laurie, MD,1 D. Neil Hayes, MD,3 James Ho, MSc,1

Janet Dancey, MD,4 Lillian L. Siu, MD1

1 Princess Margaret Hospital Phase II Consortium, Toronto, Canada. E-mail: [email protected] University of Chicago Phase II Consortium, Chicago, Illinois3 The University of North Carolina, Chapel Hill, North Carolina4National Cancer Institute, Bethesda, Maryland

Accepted 6 October 2008Published online 23 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hed.21052

Abstract: Background. Gene amplification status of theepidermal growth factor receptor (EGFR) and the human epi-dermal growth factor receptor 2 (HER2) were analyzed andcorrelated with clinical outcome in patients with progressivemalignant salivary glands tumors (MSGT) treated with the dualEGFR/Her2 tyrosine kinase inhibitor lapatinib.

Methods. Fluorescence in situ hybridization (FISH) analysis

for both EGFR and HER2 gene amplification was performed

successfully in the archival tumor specimens of 20 patients

with adenoid cystic carcinomas (ACC) and 17 patients with

non-ACC, all treated with lapatinib.

Results. For ACC, no EGFR or HER2 amplifications were

detected. For non-ACC, no EGFR gene amplifications were

detected but 3 patients (18%) were HER2 amplified and all had

stained 3þ for both EGFR and HER2 by immunohistochemistry

(IHC) in their archival specimens. Two of these patients had

time-to-progression (TTP) durations of 8.3 months and 18.4

months, respectively. Interestingly, patients with low and high

HER2/chromosome-specific centromeric enumeration probe

(CEP) 17 ratio had a prolonged TTP than those with moderate

ratios for both ACC and non-AAC subtypes.

Conclusions. HER2 to CEP17 FISH ratio may predict which

patients with MSGT have an increased likelihood to benefit from

lapatinib. The finding of HER2:CEP17 ratio as a predictive marker

of efficacy to lapatinib warrants further investigation. VVC 2009

Wiley Periodicals, Inc. Head Neck 31: 1006–1012, 2009

Keywords: MSGT; lapatinib; EGFR and HER2 gene amp-

lification; FISH

Malignant salivary gland tumors (MSGT) onlyrepresent approximately 7% of all head and

Correspondence to: L. L. Siu

Contract grant sponsor: NCI; contract grant numbers: N01-CM-62203,N01-CM-57018-16; contract grant sponsor: Translational ResearchInitiative; contract grant number: 22XS108-09; contract grant sponsor:GlaxoSmithKline.

Presented in part at the 2007 AACR-NCI-EORTC International Confer-ence on Molecular Targets and Cancer Therapeutics. October 22–26,2007, San Francisco, California.

D. Neil Hayes and Roger B. Cohen have reported a financial interest/relationship with GSK as a participant on the advisory board for the de-velopment of Lapatinib, and as the recipient of research support,respectively.

VVC 2009 Wiley Periodicals, Inc.

1006 EGFR and HER2 Gene Amplification in Salivary Gland Cancer HEAD & NECK—DOI 10.1002/hed August 2009

neck tumors. Surgery and/or radiotherapy arethe primary treatment options when disease islocalized. Adenoid cystic carcinomas (ACC) con-stitute between the 30% and 40% of the majorand minor salivary gland cancers, respectively.These tumors are characterized by both late localrecurrences and distant metastases and their poorresponse to conventional cytotoxic chemotherapy.1–4

Contrarily, the nonadenoid cystic carcinomas(non-ACC) encompass a more heterogeneousgroup with different histologies, clinical presenta-tion, and biological behavior. Although non-ACCtend to have better response rates to chemother-apy agents, these are usually short-lasting.5,6

The development of new treatment strategiesfor MSGT remains a challenge, especially inpatients who have a relapse after definitive localtherapy or those with metastatic disease. Previ-ous reports indicate that MSGT present variabledegree of EGFR and/or HER2 protein overex-pression.7–11 Although the prognostic signifi-cance of EGFR overexpression has not been welldefined, overexpression of the HER2 oncoproteinhas been associated with biological aggressive-ness and poor prognosis in most studies.12,13

Therefore, there is a strong rationale to investi-gate the antitumor effect of agents targetingthese receptors in patients with MSGT. On thebasis of the clinical experience with targetedagents to EGFR and HER2 in other tumortypes, the identification of biological markers isimportant and may help to select MSGT sub-groups most likely to respond to these agents.Recent reports demonstrated that increasedEGFR gene copy number as detected by fluores-cence in situ hybridization (FISH) appears tocorrelate with improved clinical outcomes innon-small cell lung cancer and patients withhead and neck cancer receiving treatment withsmall molecule EGFR tyrosine kinase inhibi-tors.14,15 Furthermore, HER2 amplification sta-tus measured by FISH has been shown tocorrelate well with HER2 protein overexpressionby immunohistochemistry (IHC) and has similarpredictive and prognostic significance in breastcancer.16,17

Therefore, the aim of this study was todetermine the gene amplification status ofEGFR and HER2 by FISH in the archival tu-mor specimens of MSGT patients participatingin a phase II clinical trial with the reversibledual EGFR and HER2 tyrosine kinase inhibitorlapainib, and to correlate such findings withclinical outcome.18

PATIENTS AND METHODS

Clinical Study. Eligible patients with ACC andnon-ACC were treated in 2 separate cohorts in amulticenter phase II trial with lapatinib givenas 1500 mg orally once daily and continuouslyuntil disease progression, unacceptable toxicity,patient refusal, or physician’s decision to with-draw the patient.18 The primary objective of thisstudy was to determine the objective responserate of lapatinib in MSGT, as classified byRECIST criteria. Secondary objectives were toevaluate the duration of response; rate and du-ration of stable disease; progression-free, me-dian and overall survival rates, as well as safetyand tolerability of lapatinib in this population.Institutional review board approval wasobtained at all participating centers.

Tumor Material. All patients enrolled in thestudy gave written informed consent for the col-lection, storage, and analysis of formalin-fixedparaffin-embedded primary tumor diagnosticspecimens. All patient histologies were previouslyreviewed and confirmed by a blinded pathologist.For a patient to be eligible for the trial, tumorsamples must have stained at least 1þ for EGFRby immunohistochemical analysis (IHC) and/or atleast 2þ for HER2 by IHC.

IHC for EGFR and erbB2 Expression. For EGFRIHC, the mouse monoclonal EGFR antibody (clone31G7, Zymed Lab, South San Francisco, CA) wasincubated during 1 hour (1:50 dilution) with tis-sue sections and detected using routine avidin-bio-tin technique. For HER2 analysis, the sectionswere subjected to antigen retrieval by boiling incitrate buffer followed by incubation with the pri-mary antibody (rabbit antip185/Her-2, Herceptest,DAKO AS, Copenhagen, Denmark). After sectionswere washed, they were incubated for 20 minuteseach with biotinylated secondary antibody, fol-lowed by streptavidin-horseradish peroxidaseusing the Multi-Species Ultra Streptavidin Kit(Signet Laboratories, Dedham, MA).

The slides were developed for 5 minutesusing the NovaRed substrate kit (Vector Labora-tories, Burlingame, CA), and then counter-stained with Mayer’s hematoxylin. Slides werescored on a 0 to 3þ scale: 0, staining in lessthan 10% of tumor cells or no staining; 1þ, faintand partial membrane staining in �10% of tu-mor cells; 2þ, weak to moderate complete mem-brane staining in �10% of tumor cells; or 3þ,

EGFR and HER2 Gene Amplification in Salivary Gland Cancer HEAD & NECK—DOI 10.1002/hed August 2009 1007

moderate to strong complete membrane stainingin �10% of tumor cells.

FISH for EGFR and HER2 Amplification. For EGFRgene amplification, FISH assay was performedin accord with a previously published protocol.19

Enumeration of the number of locus-specificidentifier (LSI) EGFR and CEP7 signals per nu-cleus was done according to the guidelines setforth by Varella-Garcia.20 Disomy, low trisomy,high trisomy, and low polysomy were classifiedas FISH negative, whereas high polysomy andgene amplification were considered FISHpositive.

For HER2, FISH was performed with the useof the FDA approved assay PathVysion HER2/neu DNA probe kit from Vysis (Abbott Molecu-lar, Des Plaines, IL) according to the manufac-turer’s directions.

Enumeration of the number of LSI HER2/neu and CEP17 signals per nucleus for 25 tu-mor cells was done by 2 independent readers. Aspecimen was considered amplified for the HER-2/neu gene with a ratio of �2.0 and nonampli-fied with a ratio of <2.0.

Statistical Analysis. Statistical analysis wascarried out using S-plus 2000 for Windows

(Insightful Corporation, Seattle, WA). Time toprogression (TTP) and overall survival (OS)estimates were calculated using the Kaplan-Meier method and 95% confidence intervals con-structed. Exploratory analysis was performed toinvestigate whether EGFR, EGFR:CEP7 ratio,HER2 or HER2:CEP17 ratio was predictive ofTTP or OS using the log-rank test. Patientswere grouped by the median EGFR and HER2score, amplification status, and IHC category.All statistical tests were 2-sided and performedat the 0.05 level of significance.

RESULTS

EGFRandHER2GeneandProteinExpression. Thirty-seven of the 39 (94.8%) archival specimens fromthe primary tumor of the patients enrolled ontothe phase II clinical trial were successfully ana-lyzed by FISH (see Figure 1). Twenty sampleswere from patients with ACC and 17 were fromnon-ACC patients. Table 1 provides details onthe histologies, EGFR and HER2 expression byIHC, EGFR and HER2 gene amplification sta-tus, and CEP ratios by FISH.

No EGFR amplifications were found in ACCor non-ACC patients. HER2 gene amplificationusing conventional criteria (HER2:CEP17 ratio

FIGURE 1. Example of FISH images.


�2.0) were not found in ACC, but were found in3 of the 17 (18%) non-ACC patients. The tumorspecimens of these 3 patients also stained 3þ byIHC for both EGFR and HER2. Overall, 75%(15/20) and 84% (16/19) of AAC and non-ACCpatients had �2þ EGFR staining by IHC,respectively. However, the frequency of HER2protein overexpression was in general low withstaining �2þ in 5% (1 of 20) of ACC and in 42%(8 of 19) of non-AAC patients. Only 5% (1 of 20)of the ACC patients and 40% (8 of 19) of thenon-AAC had any positive staining for bothEGFR/HER2.

Association with Clinical Outcome. The clinicaloutcome of the phase II trial has previously beenreported.18 No objective responses were seen but9 of 20 ACC patients and 4 of 19 non-ACCpatients (total 36%) had stabilization of diseasefor more than 6 months. For these patients,the mean TTP before initiating therapy was3 months (range, 2–6).18 These patients with bet-ter clinical outcome had greater frequencies of3þ EGFR (46%) or 3þ HER2 (75%) expressionby IHC. As mentioned, 3 of non-AAC patientswere HER2 amplified. Only 2 of them wereevaluable for efficacy. These 2 patients who had

Table 1. IHC and FISH results of ACC and non-ACC patients for EGFR and HER2.

EGFR

FISH

HER2

FISH

EGFR

IHC

HER2

IHC

HER2:CEP17

ratio

HER2 FISH

signal

EGFR:CEP7

ratio

EGFR FISH

signal Histology

TPP

(d)

Non-Amp Non-Amp 1þ 1þ 1.00 55 1.2 Undifferentiated carcinoma 162

Non-Amp Non-Amp 2þ 3þ 1.00 58 1.1 57 Salivary duct carcinoma 83

Failed Failed 3þ 0 Adenocarcinoma 104


Non-Amp Non-Amp 3þ 0 0.96 53 1.1 59 Salivary duct carcinoma NE

Non-Amp Non-Amp 2þ 0 1.10 62 0.90 56 Squamous cell carcinoma 112

Non-Amp Amplified 3þ 3þ 4.60 463 1.1 69 Adenocarcinoma 559

Non-Amp Non-Amp 2þ 0 1.10 52 1.0 47 Mucoepidermoid 55

Non-Amp Amplified 3þ 3þ 4.00 201 0.96 28 Undifferentiated carcinoma 251

Non-Amp Amplified 3þ 3þ 3.00 228 1.1 65 Squamous carcinoma NE

Failed Failed 3þ 2þ Adenocarcinoma 7

Non-Amp Non-Amp 3þ 0 0.90 57 1.1 53 Squamous carcinoma 56

Non-Amp Non-Amp 3þ 0 1.00 52 1.2 52 Undifferentiated carcinoma 50

Non-Amp Non-Amp 1þ 1þ 0.92 59 1.1 53 adenocarcinoma 41


Failed Failed 2þ 1þ Acinic cell 49

Non-Amp Non-Amp 3þ 0 1.00 58 0.98 43 Mucoepidermoid 28

Non-Amp Non-Amp 3þ 2þ 1.00 48 1.1 44 Adenocarcinoma 49

Non-Amp Non-Amp ? ? 0.96 53 1.0 94 Adenocarcinoma 65

Failed Non-Amp 3þ 3þ 0.86 56 Adenocarcinoma 287

ACC

Non-Amp Non-Amp 2þ 0 1.20 77 0.92 62 650

Non-Amp Non-Amp 1þ 0 1.00 67 1.0 59 112

Non-Amp Non-Amp 3þ 1þ 0.95 58 1.1 50 202

Non-Amp Non-Amp 3þ 1þ 1.10 76 0.89 55 587

Non-Amp Non-Amp 2þ 0 1.00 59 0.98 57 79

Non-Amp Non-Amp 1þ 0 1.00 54 0.91 43 7

Non-Amp Non-Amp 1þ 0 0.90 64 0.85 47 107

Non-Amp Non-Amp 2þ 1þ 1.10 57 1.0 54 109

Non-Amp Non-Amp 1þ 0 0.81 63 0.95 61 531

Non-Amp Non-Amp 2þ 1þ 1.10 60 1.0 56 221

Non-Amp Non-Amp 2þ 0 0.83 54 0.82 51 55

Non-Amp Non-Amp 2þ 2þ 1.00 53 1.1 57 24

Non-Amp Non-Amp 2þ 0 0.93 52 1.0 49 93

Non-Amp Non-Amp 2þ 0 0.91 58 1.1 46 385

Non-Amp Non-Amp 2þ 0 1.00 58 0.98 43 94

Non-Amp Non-Amp 2þ 1þ 0.95 63 0.90 53 12

Non-Amp Non-Amp 3þ 0 0.96 70 1.1 52 111

Non-Amp Non-Amp 1þ 0 1.00 71 1.1 73 104

Non-Amp Non-Amp 3þ 0 0.86 45 1.0 56 356

Non-Amp Non-Amp 3þ 1þ 0.90 53 0.85 47 64

Note: Two non-ACC patients not evaluable (NE) due to (1) patient had prior history of second malignancy and (2) patient did not receive study drug.


poorly differentiated histologies (1 adenocarci-noma, 1 undifferentiated carcinoma), achieved 2of the longest durations of TTP with 18.4 monthsand 8.3 months, respectively. The median follow-up duration for all patients was 15.8 months.

Exploratory analyses were performed usingthe EGFR:CEP7 and HER2:CEP17 ratios as pre-dictors for TTP and OS in 20 ACC patients and16 non-ACC patients whose FISH and efficacyoutcome data were both available. No significantcorrelations were found between the EGFR:CEP7ratio and clinical outcome. In an exploratoryattempt to correlate the HER2:CEP17 ratio withclinical outcome, different cut-off values of the ra-tio were utilized beyond the conventional amplifi-cation definition. For instance, when ACC andnon-ACC patients were classified based on thecut-off value for the HER2:CEP17 ratio of lessthan 1 versus 1 or greater, no statistically signifi-cant differences were found between the sub-groups (see Figure 2). However, using anarbitrary set of cut-off values to define theHER2:CEP17 ratio as low (�0.91), moderate(0.92–1.09) or high (�1.10), a more striking sepa-ration in outcome was observed between the sub-groups. Of note, 0.91 was selected since it isequivalent to a ratio of 1:1.10. Significant differ-ences in the clinical outcome of these patientswas observed (Table 2). For ACC patients, theTTP of patients with low/high ratios was9.5 months versus 3.1 months for those withmoderate ratios (p ¼ .03). For non-ACC patients,the TTP of patients with low/high ratios was8.3 months versus 1.6 months for those with mod-erate ratios (p ¼ .015) (see Figure 3). Differenceswere also detected in the OS of these subgroupsalthough not statistically significant (Table 2).

DISCUSSION

The identification of predictive markers remainsa challenge in this era of novel molecularly tar-geted therapeutics. Correlative studies havebecome an integral part of clinical trials to es-tablish relevant predictors of response to tar-geted therapies. Our study evaluated the valueof EGFR and HER2 gene amplification in pre-dicting outcome for patients with MSGT treatedwith lapatinib. This is the first study to analyzeEGFR and HER2 gene amplification by FISHand to correlate with clinical outcome in thisuncommon tumor type. These molecular abnor-malities may confer different disease behaviorand determine response to targeted therapies.

No EGFR gene amplifications were found ineither ACC or non-ACC despite the high propor-tion of patients with �2þ EGFR protein expres-sion by IHC. Although significant discrepanciesbetween publications have been observed, the

FIGURE 2. Time to progression for ACC and non-ACC patients classified based on HER2:CEP17 ratio of <1 versus �1.

Table 2. Median TTP and OS for ACC and non-ACC patients

based on HER2 to CEP17 ratio—an exploratory analysis.

TTP Non-ACC

p

value ACC

p

value

Her2 <1 1.89 (1.5-NR) .69 3.59 (2.11-NR) .93

Her2 >¼1 2.73 (1.64-NR) 3.50 (2.60-NR)

Moderate 1.64 (1.48-NR) .015 3.08 (0.79-NR) .032

Low/high

expression

8.26 (3.68-NR) 9.49 (3.52-NR)

OS

Her2 <1 7.75 (3.06-NR) .11 NR .65

Her2 >¼1 21.27 (7.34-NR) NR

Moderate 9.11 (6.38-NR) .17 NR .49

Low/high

expression

NR NR


frequency of EGFR protein overexpression foundin our patients with MSGT was similar to previ-ous reports.7–9,12,21 Not surprisingly, EGFR pro-tein expression did not correlate with tumorresponse. Most previous studies have shownthat expression of EGFR protein in archivaltumors by IHC is an unreliable predictor ofresponsiveness to EGFR inhibitor agents.22,23

In our study, no HER2 gene amplificationswere found in ACC patients. Although only fewHER2 gene amplifications were found in non-AAC patients, these patients had better out-comes with lapatinib. The HER2 amplifiedpatients all had 3þ EGFR and HER2 stainingby IHC, suggesting that HER2 amplification sta-tus by FISH based on conventional criteria didnot contribute additional predictive or prognos-tic value in our study. Our findings are consist-ent with the general observation that EFGR butnot HER2 seems to be greater expressed in ACCand vice versa in non-ACC.24 In our study, 9 of39 (23%) patients with MSGT presented with2þ and 3þ HER2 protein expression and higherfrequency was observed for non-ACC patients (8of 19, 42%). Among the 8 non-ACC tumors over-expressing HER2, the histologies were as fol-lows: adenocarcinomas (n ¼ 4), salivary ductcarcinomas (n ¼ 2), undifferentiated carcinoma(n ¼ 1), and squamous cell carcinoma (n ¼ 1).Recent data indicate that some histologies ofMSGT, in particular salivary duct carcinomas,present with higher rates of HER2 positivity ei-ther by IHC and FISH,25,26 and may be morelikely to benefit from agents targeting HER2such as trastuzumab.27 Furthermore, a parallelcan be drawn between our findings in MSGT

and that reported within patients with meta-static breast cancer treated with lapatinib.28,29

In the latter, HER2 overexpression measuredqualitatively or quantitatively was more predic-tive of response with lapatinib than EGFRexpression.

From these results, the benefit of addingHER2 gene amplification by FISH as a predic-tive marker for response to lapatinib seems verylimited in MSGT. However, we did observe aninteresting relationship between HER2:CEP17FISH ratio and clinical outcome for both ACCand non-ACC when a nonconventional set of cri-teria was used. The conventional criteria ofHER2:CEP17 � 2 as a cut-off to define amplifi-cation status in breast cancer does not necessar-ily apply to MSGT, as these malignancies likelypossess different disease biology. In our explora-tory analysis, patients were grouped arbitrarilyinto moderate versus low/high groups, based onnon-conventional cut-off values for the HER2:CEP17 ratio. This subgroup evaluation,intended to be hypothesis generating, may haveyielded a relevant marker for MSGT patientstreated with agents targeting HER2. Patientswith low or high HER2:CEP17 ratios appear tohave a longer TTP than those with moderateratios, reflecting improved clinical outcomes. Alow HER2:CEP17 ratio may have prognosticvalue and reflects patients who have a lessaggressive disease biology, whereas a highHER2:CEP17 ratio may have predictive valueand reflects patients who benefited most fromlapatinib. This finding needs to be confirmedwith future larger studies in this disease.Although designing trials to validate these

FIGURE 3. Time to progression for ACC and non-ACC patients classified based on low/high versus moderate HER2:CEP17 ratios.


differences is challenging because of the smallnumber of MSGT cases, recent studies have pro-ven that, through collaborative group efforts,accrual can be rapid and optimized.18,30

Acknowledgments. The authors acknowledgeKevin Jackson and Nigel Biswas-Baldwin fortheir assistance with this project.

REFERENCES

1. Spiro RH. Distant metastases in adenoid cystic carci-noma of the minor salivary glands. Am J Surg 1997;174:495–498.

2. Mattox DE, Von Hoff DD, Balcerzak SP, et al. Southwestoncology group study of mitoxantrone for treatment ofpatients with advanced adenoid cystic carcinoma of thehead and neck. Invest New Drugs 1990;8:105–107.

3. Vermorken JB, Verweij J, de Mulder PH, et al. Epirubi-cin in patients with advanced or recurrent adenoid cysticcarcinoma of the head and neck: a phase II study of theEORTC Head and Neck Cancer Cooperative Group. AnnOncol 1993;4:785–788.

4. Verweij J, de mUlder PH, de Graeff A, et al. Phase IIstudy on mitoxantrone in adenoid cystic carcinomas ofthe head and neck. EORTC Head and Neck Cancer Co-operative Group. Ann Oncol 1996;7:867–869.

5. Goode RK, Auclair Pl, Ellis GL. Mucoepidermoid carci-noma of the major salivary glands: clinical and histopa-thologic analysis of 234 cases with evaluation of gradingcriteria. Cancer 1998;82:1217–1224.

6. Dimery IW, Legha SS, Shirinian M, Hong WK. Fluorour-acil, doxorubicin, cyclophosphamide, and cisplatin combi-nation chemotherapy in advanced or recurrent salivarygland carcinoma. J Clin Oncol 1990;8:1056–1062.

7. Chen CH, Li BY, Wan JT, Sun A, Leu JS, Chiang CP.Expression of epidermal growth factor in salivaryadenoid cystic carcinoma. Proc Natl Sci Counc RepubChina B 2001;25:90–106.

8. Shintani S, Funayama T, Yoshihama Y, et al. Expressionof c-erbB family gene products in adenoid cystic carci-noma of salivary glands: an immunohistochemical study.Anticancer Res 1995;15:2623–2626.

9. Vered M, Braunstein E, Buchner A. Immunohistochemi-cal study of epidermal growth factor receptor in adenoidcystic carcinoma of salivary gland origin. Head Neck2002;24:632–636.

10. Cho KJ, Lee SS, Lee YS. Proliferating cell nuclear anti-gen and c-erbB-2 oncoprotein expression in adenoidcystic carcinomas of the salivary glands. Head Neck1999;21:414–419.

11. Skalova A, Starek I, Vanecek T, et al. Expression ofHER-2/neu gene and protein in salivary duct carcinomasof parotid gland as revealed by fluorescence in-situhybridization and immunohistochemistry. Histopathology2003;42:348–356.

12. Press MF, Pike MC, Hung J, et al. Amplification andoverexpression of HER-2/neu in carcinomas of the sali-vary gland: correlation with poor prognosis. Cancer Res1994;54:5675–5682.

13. Sugano S, Mukai K, Tsuda H, et al. Immunohistochemi-cal study of c-erbB-2 oncoprotein overexpression inhuman major salivary gland carcinoma: an indicator ofaggressiveness. Laryngoscope 1992;102:923–927.

14. Cappuzzo F, Varella-Garcia M, Shigematsu H, et al.Increased HER2 gene copy number is associated withresponse to gefitinib therapy in epidermal growth factor

receptor-positive non-small-cell lung cancer patients.J Clin Oncol 2005;23:5007–5018.

15. Agulnik M, da Cunha Santos G, Hedley D, et al. Predic-tive and pharmacodynamic biomarker studies in tumorand skin tissue samples of patients with recurrent or met-astatic squamous cell carcinoma of the head and necktreated with erlotinib. J Clin Oncol 2007;25:2184–2190.

16. Jacobs TW, Gown AM, Yaziji HC, Barnes MJ, SchnittSJ. Comparison of fluorescence in situ hybridization andimmunohistochemistry for the evaluation of HER-2/neuin breast cancer. J Clin Oncol 1999;17:1974–1982.

17. Jimenez RE, Wallis T, Tabasczka P, Wisscher DW. Deter-mination of Her-2/Neu status in breast carcinoma: compar-ative analysis of immunohistochemistry and fluorescent insitu hybridization. Mol Pathol 2000;13:37–45.

18. Agulnik M, Cohen EW, Cohen RB, et al. Phase II studyof lapatinib in recurrent or metastatic epidermal growthfactor receptor and/or erbB2 expressing adenoid cysticcarcinoma and non adenoid cystic carcinoma malignanttumors of the salivary glands. J Clin Oncol 2007;25:3978–3984.

19. Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al. Epider-mal growth factor receptor in non-small cell lung cancercarcinomas: correlation between gene copy number andprotein expression and impact on prognosis. J Clin Oncol2003;21:3798–3807.

20. Varella-Garcia M. Stratification of non-small cell lungcancer patients for therapy with epidermal growth factorreceptor inhibitors; the EGFR fluorescence in situhybridization assay. Diagn Pathol 2006;1:19–29.

21. Yamada K, Iwai K, Okada Y, et al. Immunohistochemicalexpression of epidermal growth factor receptor in sali-vary gland tumours. Virchows Arch A Pathol Anat His-topathol 1989;415:523–531.

22. Dancey JE. Predictive factors for epidermal growth fac-tor receptor inhibitors: The bull’s-eye hits the arrow.Cancer Cells 2004;5:411–415.

23. Perez-Soler R, Chachoua A, Hammond LA, et al. Deter-minants of tumor response and survival with erlotinib inpatients with non-small cell lung cancer. J Clin Oncol2004;22:3238–3247.

24. Laurie SA, Licitra L. Systemic therapy in the palliativemanagement of advanced salivary gland cancers. J ClinOncol 2006;24:2673–2678.

25. Glisson B, Colevas AD, Haddad R, et al. HER2 expres-sion in salivary gland carcinomas: dependence on histo-logical subtype. Clin Cancer Res 2004;10:944–946.

26. Dagrada GP, Negri T, Tamborini E, Pierotti MA, PilottiS. Expression of HER-2/neu gene and protein in salivaryduct carcinomas of parotid gland as revealed by fluores-cence in-situ hybridization and immunohistochemistry.Histopathology 2004;44:301–302.

27. Haddad R, Colevas AD, Krane JF, et al. Herceptin inpatients with advanced or metastatic salivary gland car-cinomas. A phase II study. Oral Oncol 2003;39:724–727.

28. Burris HA III, Hurwitz HI, Dees EC, et al. Phase Isafety, pharmacokinetics, and clinical activity study oflapatinib (GW572016), a reversible dual inhibitor of epi-dermal growth factor receptor tyrosine kinases, in heav-ily pretreated patients with metastatic carcinomas.J Clin Oncol 2005;23:5305–5313.

29. Spector NL, Xia W, Burris HA III, et al. Study of the bio-logic effects of lapatinib, a reversible inhibitor of ErbB1and ErbB2 tyrosine kinases, on tumor growth and sur-vival pathways in patients with advanced malignancies.J Clin Oncol 2005;23:2502–2512.

30. Hotte SJ, Winquist EW, Lamont E, et al. Imatinib mesy-late in patients with adenoid cystic cancers of the sali-vary glands expressing c-kit: a Princess MargaretHospital phase II consortium study. J Clin Oncol 2005;23:585–590.


fluorescence in situ hybridization gene amplification analysis of egfr and her2 in patients with...

Documents