Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance

Published OnlineFirst April 14, 2010; DOI: 10.1158/1078-0432.CCR-09-0238

Clinical Human Cancer Biology Cancer Research Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance

Michelle D. Williams1, Dianna B. Roberts2, Merrill S. Kies3, Li Mao3, Randal S. Weber2, and Adel K. El-Naggar1,2

Abstract Purpose: Salivary duct carcinoma overexpresses epidermal growth factor receptor (EGFR) and HER-2, although the underlying mechanisms remain undefined. Because of the potential utilization of these markers as treatment targets, we evaluated protein and gene status by several techniques to determine complementary value. Experimental Design: A tissue microarray of 66 salivary duct carcinomas was used for immunohistochemical analysis of HER-2 and EGFR expression (semiquantitatively evaluated into a three-tiered system), and fluorescence in situ hybridization for gene copy number, and chromosomes 7 and 17 ploidy status. Sequencing of exons 18, 19, and 21 of the EGFR gene for mutations was carried out. Result: For EGFR, 46 (69.7%) of the 66 tumors showed some form of EGFR expression (17 at 3+, 17 at 2+, 12 at 1+) but none gene amplification. Five (9.4%) of 53 tumors showed mutations in exon 18 (n = 3) and exon 19 (n = 2). Polysomy of chromosome 7 (average >2.5 copies/cell) was detected in 15 (25.0%) of 60 tumors (6 at 3+, 5 at 2+, 2 at 1+, 2 at 0+ expression) and correlated with poor 3-year survival (P = 0.015). For HER-2, 17 (25.8%) of 66 tumors expressed HER-2 (10 at 3+, 3 at 2+, 4 at 1+). Eight tumors showed HER-2 gene amplification (6 at 3+, 1 at 1+, 1 at 0+ protein expression). Chro- mosome 17 polysomy was found in 8 (15.7%) of 51 tumors; two had HER-2 expression (3+, 1+). Conclusion: Our study shows that salivary duct carcinomas (a) harbor EGFR gene mutations in a subset of tumors that may guide therapy, (b) pursue an aggressive clinical course in cases with chromosome 7 polysomy and high EGFR expression, and (c) with HER-2 gene amplification and protein high expression, may be selected for targeted therapy. Clin Cancer Res; 16(8); 2266–74. ©2010 AACR.

Salivary duct carcinoma is a high-grade, clinically ag- hormonal and growth factors that are known to play a cen- gressive adenocarcinoma variant with remarkable pheno- tral role in the biology of breast cancers, including HER2 typic and biological resemblance to mammary high-grade and epidermal growth factor receptor (EGFR; refs. 5, 8). ductal carcinoma (Figs. 1A and 1C; refs. 1, 2). Patients However, the optimum methodology that best reflects with locally advanced, recurrent, and/or metastatic disease their functional level for therapeutic purposes remains a have limited therapeutic options and generally succumb to subject of debate. their disease. In the past two decades several individual Growth factors and their receptors play a central role in genes, markers of different cellular pathways, chromo- mammary ductal tumorigenesis and in other adenocarci- some alterations, and hormonal and growth factor nomas, but the expression levels alone fail to predict res- receptors have been evaluated in an effort to define the ponders to therapies (9). Several studies have shown that underlying molecular and biological pathways associated HER-2 gene amplification status has improved the identi- with their development and progression (1, 3–7). Several fication of possible responders to targeted therapy with of these studies have led to the characterization of certain trastuzumab (10–14). However, concurrent expression and gene amplification studies have identified a subset Authors' Affiliations: Departments of 1Pathology, 2Head and Neck of low and intermediate HER-2 gene expressions with high Surgery, and 3Head and Neck Medical Oncology, The University of gene amplification, suggesting that immunohistochemical Texas M.D. Anderson Cancer Center, Houston, Texas analysis alone leads to underestimation of patients eligible Note: Supplementary data for this article are available at Clinical Cancer for therapy (14, 15). Central to addressing this issue is Research Online (http://clincancerres.aacrjournals.org/). whether protein expression and gene amplification are Corresponding Author: Adel K. El-Naggar, Professor of Pathology and Head and Neck Surgery, The University of Texas M.D. Anderson Cancer mutually exclusive or complementary events in assessing Center, 1515 Holcombe Boulevard, Unit 085, Houston, TX 77030. Phone: the biological role of these factors for therapeutic stratifi- 713-792-3109; Fax: 713-792-5532; E-mail: [email protected]. cation of patients with these tumors. Accordingly, initial doi: 10.1158/1078-0432.CCR-09-0238 immunohistochemical screening followed by gene ampli- ©2010 American Association for Cancer Research. fication analysis by fluorescence in situ hybridization

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EGFR and HER-2 Analysis in Salivary Duct Carcinoma

M.D. Anderson Cancer Center formed this study. The cur- Translational Relevance rent WHO guidelines were used for classifying tumors as salivary duct carcinomas (30). A tissue microarray was crea- Our concurrent analysis, the most comprehensive ted using two 1.0-mm diameter cores consisting of repre- to date, of the expression and the genomic alterations sentative tumor from each paraffin block consisting of EGFR of the epidermal growth factor receptor ( )and tumor tissue fixed in 10% buffered formalin. The tissue mi- HER-2 receptor, defines the molecular alterations of croarray was used for immunohistochemistry and FISH these genes in salivary duct carcinomas for the biolo- evaluations. Pathologic findings, which included immuno- gical stratification of patients with these tumors. Both histochemistry, gene amplification, and mutational status, EGFR activating mutations (10%) in the gene and were compared with clinical factors that included gender, chromosome 7 polysomy (25%) were identified in a age, and stage along with clinical outcomes and were eva- subset of salivary duct carcinomas. These alterations luated by Fisher's exact test and χ2 based on the number of have correlated with tyrosine kinase inhibitor treat- comparative groups. A P value of 0.05 was considered ment response in different adenocarcinomas and significant. may potentially be applicable in salivary duct carcinoma patients. Additionally, chromosome 7 polysomy Immunohistochemical analysis portends a poorer clinical outcome that trends with Immunohistochemical analysis for HER-2 and EGFR high EGFR expression. The finding of both overexpres- was done using the automated BOND MAX immunohis- sion of HER-2 and gene amplification defines a group tochemistry stainer by Vision Biosystems on 4-μm paraf- of patients who may benefit from targeted therapy fin sections of the tissue microarray material. In brief, with trastuzumab. The results of this study highlight following dewaxing, washing, and rehydration of the the spectrum of alterations in the EGFR family of slides through xylene and graded alcohols, Tris-EDTA receptors as potential targets for therapy in salivary buffer was used for antigen retrieval. Slides were subse- duct carcinomas. quently treated with 3% hydrogen peroxide to block endogenous peroxidase. Following incubation with the primary antibodies, HER-2 (clone e2-4001, mouse, (FISH) has been recommended for borderline cases in 1:300, Labvision) and EGFR (clone 31G7, mouse, 1:50, mammary carcinomas (14, 16). The correlation between Zymed), the secondary conjugate antibody was applied. the immunohistochemical expression and the genomic Finally, each specimen-containing slide was developed ′ status of the HER-2 gene in salivary duct carcinoma re- using the chromogen 3,3 -diaminobenzidine and coun- mains poorly defined secondary to the limited number terstained with hematoxylin. of tumors studied to date (14, 17–20). Immunohistochemically stained tumor sections for Recently, mutational analysis of the EGFR gene has HER-2 were evaluated for membranous expression: 3+, identified a subset of non–small cell lung carcinomas that strong complete in >10%; 2+, weak complete in >10%; responded to tyrosine kinase inhibitors (TKI; refs. 21–23). 1+, partial staining of tumor cells in >10%; 0, negative Other molecular alterations, including chromosomal or <10% staining. EGFR immunohistochemistry in tumor polysomy, may also play a role in the pathogenesis asso- cells was evaluated for membranous expression: 3+, strong ciated with this pathway (24–28). Because at least half of membrane staining in >10%; 2+, moderate membranous salivary duct carcinomas express EGFR (5, 29) and little is staining in >10%; 1+, weak membranous staining in known on the status of the EGFR expression, correlative >10%, 0, negative or <10% staining. assessment of these findings may determine the association between expression and underlying molecular factors, FISH μ including mutational and gene amplification status. A4- m paraffin section of the tissue microarray was Our objectives were to concurrently analyze, for the first analyzed by FISH using the manufacturer's recommended time, the expression and the genomic status of EGFR and standard methods for HER-2 [PathVysion HER-2 DNA HER-2 genes in a large cohort of salivary duct carcinomas. Probe Kit; HER-2 Spectrum Orange/centromere enhancer We used immunohistochemical analysis and FISH to de- of position effect (CEP)17 Spectrum Green, Vysis, Abbott] termine their relationship in the biological stratification of and EGFR (Vysis LSI EGFR Spectrum Orange/CEP 7 Spec- patients with salivary duct carcinoma. We also determined trum Green, Vysis, Abbott). A known HER-2 amplified the incidence and nature of biomarkers that may guide tar- ductal adenocarcinoma was included in the tissue micro- geted therapies in a subset of patients with salivary duct array as a positive control as well as normal salivary tissue carcinoma. as a negative control. Each tumor sample was evaluated for the signals of HER- 2 or EGFR signal and amplification pattern (spectrum or- Materials and Methods ange) and the number of centromeric probe signals to chromosome 7 or 17 (spectrum green), respectively, within Sixty-six salivary duct carcinomas with archival formalin- at least 20 tumor cells evaluated from both cores. Amplifi- fixed paraffin blocks available at the University of Texas, cation was defined as clusters of probes (>10 copies/tumor

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cell) documented in ≥20% of cells analyzed. Tumors were Although EGFR expression correlated significantly with also evaluated for low levels of amplification by comparing local recurrence (34.6% versus 65.4%, P = 0.046) and was the gene to centromeric signals as a ratio, with a ratio <1.8 more frequently associated with distant metastases (44.0% defined as nonamplified, 1.8 to 2.2 as indeterminate, and versus 56.0%) and poor 3-year survival (59.1% versus >2.2 as amplified (14). Additionally, chromosome copy 40.9%), it did not reach statistical significance (P =0.6 number for chromosomes 7 and 17 were evaluated. Tu- and 0.5, respectively). mors were classified based on the percentage of cells with Gene amplification(FISH). EGFR gene amplification was increased chromosomal numbers as outlined by Cappuzzo not identified in any of the 66 salivary duct carcinomas et al. (31), as well as the average number of centromeres analyzed by FISH. per cell with evaluation for correlation with clinical and Chromosomal ploidy. An increase in the chromosome 7 pathologic parameters. Chromosomal copy numbers per copy number in tumor cells (average >2.5 copies/cell) tumor cell were classified as the following: disomy, ≤2 cop- was found in 15 (25.0%) of the 60 tumors suitable for ies in ≥90% of cells; low trisomy, 3 copies in <40% of cells; analysis (Table 2, Fig. 1B). Tumors with an average ploi- high trisomy, 3 copies in ≥40% of cells; low polysomy, ≥4 dy of >2.5 copies for chromosome 7 were significantly copies in <40% of cells; and high polysomy, ≥4 copies in correlated with a lower 3-year survival (P = 0.002) and ≥40% of tumor cells (31). difference in overall survival by Kaplan-Meier assessment (P = 0.015; Fig. 2). Categorical classification showed 13 EGFR gene sequence mutational analysis (21.7%) tumors with disomy for chromosome 7, 25 DNA from paraffin-embedded formalin-fixed tumor (41.7%) as low trisomy, 16 (26.7%) as low polysomy, was enriched to >70% tumor cells and was suitable for se- 0 (0.0%) as high trisomy, and 6 (10.0%) as high poly- quencing exons 18, 19, and 21, hotspots of the EGFR gene somy (Table 2). Correlative analysis using the Kaplan- in 53 of the 66 tumors. PCR was done using previously Meier method showed a decline in median survival with described primers (27). Amplified target DNA was then se- increased chromosomal alterations from 52.0 months for quenced using ABI Big DyeTMv3.1 dye terminator cycle se- chromosome 7 disomy to 37.0 months for low trisomy, quencing. Forward and reverse sequencing was carried out. 30.5 months for low polysomy, and 14.5 months for A lung adenocarcinoma with known EGFR gene mutation chromosome 7 high polysomy (Table 2, Fig. 3). All pa- was used as a positive control. tients alive without disease were noted to be diploid for chromosome 7. Salivary duct carcinomas with increased chromosome 7 copy number (>2.5 average) showed a Results more rapid clinical course with median survival of 20.0 versus 43.0 months for chromosome 7 <2.5 (P = 0.015; EGFR Table 2). Immunohistochemistry. Forty-six (69.7%) of 66 salivary Chromosome 7 polysomy was present in 13 (28.3%) of duct carcinomas showed membranous expression for the 46 tumors with EGFR expression (Table 1). Specifically, EGFR. Of the positive tumors, 17 (37.0%) showed strong polysomy was noted in 2 (11.8%) of 0+, 2 (16.7%) of 1+, complete membranous staining (3+), 17 (37.0%) were in- 5 (31.3%) of 2+, and 6 (40.0%) of 3+ tumors, although termediate (2+), and 12 (26.0%) had weak membranous this association did not quite reach statistical significance staining (1+; Table 1). (P =0.06).

Table 1. Comparative methods for analyzing EGFR and HER-2 in salivary duct carcinoma

Category Immunohistochemistry expression score Total 01+2+ 3+

EGFR IHC 20 (30.3%) 12 (18.2%) 17 (25.8%) 17 (25.8%) 66 Gene amp 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) Polysomy* chr 7 2/17 (11.8%) 2/12 (16.7%) 5/16 (31.3%) 6/15 (40.0%) 15/60 (25.0%) HER-2 IHC 49 (74.2%) 4 (6.1%) 3 (4.5%) 10 (15.2%) 66 Gene amp 1† (2.0%) 1 (25.0%) 0 (0.0%) 6 (60.0%) 8 (12.1%) Polysomy* chr 17 6/36 (16.7%) 1/4 (25.0%) 0/3 (0.0%) 1/8 (12.5%) 8/51 (15.7%)

Abbreviations: chr, chromosome; Gene amp, gene amplification, IHC, immunohistochemistry. *Polysomy of corresponding chromosome, average copy number >2.5 †This HER-2–amplified tumor showed 3+ immunohistochemistry on whole tissue section

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EGFR and HER-2 Analysis in Salivary Duct Carcinoma

Table 2. Assessment of polysomy using average chromosome number versus categorical classification

Chr Average chr no. Categorical classification* ≥2.5 ≥3.0 Disomy Low trisomy High trisomy Low polysomy High polysomy ≥90% <40% ≥40% <40% ≥40%

17 (n = 51) 8 (15.7%) 4 (7.8%) 23 (45.1%) 10 (19.6%) 1 (2.0%) 12 (23.5%) 5 (9.8%) 7(n = 60) 15 (25%) 10 (16.7%) 13 (21.7%) 25 (41.7%) 0 (0.0%) 16 (26.7%) 6 (10%) Median survival (mo)† Chr 7 20.0 vs. 22.0 vs. 52 37 na 30.5 14.5 (43.0 for <2.5) (37.0 for <3.0)

Abbreviation: na, not applicable. *Disomy defined as ≤2 chromosome copies in >90% of tumor cells; low trisomy, 3 chromosomes in <40% of cells; high trisomy, 3 chromosomes in ≥40% of cells; low polysomy, ≥4 chromosomes in <40% of cells; high polysomy, ≥4 chromosomes in ≥40% of cells. †Median survival from Kaplan-Meier analyses (Figs. 2, 3).

Mutational analysis. Our mutational analysis, the first EGFR expression by immunohistochemistry was present in these tumors, showed that 5 (9.4%) of the 53 tumor in four of the five mutated tumors; two 3+, one 2+, and specimens suitable for this analysis had mutations in exons one 1+ expression. Polysomy of chromosome 7 was 18 and 19, hot spots of the EGFR gene: three in exon 18 present in one of the three cases evaluable by FISH. HER-2 and two in exon 19 (Supplementary Fig. S1). Point muta- was not expressed or amplified in any of these five tumors. tions in exon 18 were identified in codons L688P, A698T, All five patients presented with advanced disease (stage III and L718P. A 15 bp in-frame deletion, E746_A750del, and or IV), and four of the five died from disease from 7 to a point mutation in codon S752P were identified in exon 42 months following diagnosis; all four developed lymph 19. No mutations were identified in exon 21 (Table 3). node or distant metastases.

Fig. 1. Two salivary duct carcinomas with chromosome 7 polysomy (A, B) and HER-2 amplification (C, D). A, C, H&E sections (×10) of ductal growth pattern. B, strong EGFR membranous expression by immunohistochemistry. Inset, FISH evaluation; green probe, polysomy of chromosome 7, with increased number/nuclei; red probe, EGFR gene, with similar increased number as chromosomal probes (on average 4 copies/nucleus); 4′, 6-diamidino-2-phenylindole (DAPI) stain highlights nuclei (blue). D, strong complete HER-2 membranous expression by immunohistochemistry. Inset, FISH evaluation; green probe, chromosome 17, generally showing two copies/nucleus; red probe, HER-2 gene amplification, showing clusters of probe corresponding to marked gene amplification; DAPI stain highlights nuclei (blue).

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Fig. 2. Kaplan-Meier curve of overall survival of salivary duct carcinoma patients by the average number of copies of chromosome 7.

HER-2 (N) stage, or clinical outcome were found (Supplementary Immunohistochemistry. Seventeen (25.8%) of the 66 Table S1). salivary duct carcinomas were positive for membranous Gene amplification (FISH). HER-2 gene amplification by HER-2 staining (Table 1). Of these, strong continuous FISH was present in 8 (12.1%) of the 66 tumors. Seven of membrane expression (3+) was present in 10 (58.8%), the eight amplified tumors showed protein expression: 6 weak continuous staining (2+) in 3 (17.6%), and weak (66.7%) with strong immunostaining (3+) and 1 with partial staining in 4 (23.5%). weak staining (1+; Table 1). The gene-amplified tumor No correlation between HER-2 expression levels and with negative immunohistochemistry expression on tissue clinical factors including age, sex, tumor (T) and node array showed areas of strong (3+) expression on the

Fig. 3. Kaplan-Meier curve of overall survival of salivary duct carcinoma patients by categorical classification based on the number of copies of chromosome 7.

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EGFR and HER-2 Analysis in Salivary Duct Carcinoma

Table 3. EGFR gene mutations identified in salivary duct carcinomas

Case Age Gender Race Size Stage LN Outcome/ Exon Type of Nucleotide PC EGFR/ Chr 7 copy # (y) (cm) status FU (mo) mutation base sub. IHC

† S54 61 M W 3 T4b N2b DOD (7) 18 PM 2063T

S22 57 F W 3 T3 N0 L-NED 18 PM 2153T

S36 73 M W 4 T4a N2b DOD (9) 18 PM 2092G

S8 41 M W 2 T3 N2b DOD (13) 19 PM 2236_2250del E746 3na A750del*

S4 58 F W >4.0 T4a N0 DOD (42) 19 MD 2254T

NOTE: All tumors arose in the parotid. Abbreviations: Chr, chromosome; DOD, dead of disease; F, female; FU, follow-up/month; IHC, immunohistochemical expression; L-NED, living no evidence of disease; M, male; MD, microdeletion; mo, month; na, not available; PM, point mutation; sub., substitution; W, white. *Mutations also identified in lung adenocarcinomas. †3+ EGFR expression on whole tissue section. corresponding whole tissue section (heterogeneous distri- EGFR and HER-2 receptors revealed shared and variable bution). HER-2 gene amplification was characterized by manifestation of these genes in salivary duct carcinomas. an excess of 5-fold (10+ copy number) per cell showing In addition, the mutational (the first in this entity), ex- clusters of probe signal (Fig. 1D). There was a significantly pression, and amplification screening together with chro- higher proportion of gene amplified cases in the 3+ immu- mosome 7 ploidy analysis provides broad genomic and nohistochemistry expression group than in the tumors phenotypic account of the EGFR alterations in this enti- with low expression (P = 0.0004). ty. The results show a trend for association between high Chromosomal ploidy. Chromosome 17 polysomy, an EGFR expression and chromosome 7 polysomy and average of >2.5 copies/cell, was present in 8 (15.7%) of poor clinical outcome, but this did not reach statistical 51 tumors; of these eight, two were HER-2 positive by significance (29, 32). In this study, the lack of correla- immunohistochemistry (one 3+ and one 1+) and one tion between EGFR gene amplification and protein ex- had HER-2 gene amplification. Ploidy classification pression suggests that alternative mechanisms including showed 23 (45.1%) tumors with chromosome 17 dis- posttranslational modification, or decreased degradation omy, 10 (19.6%) low trisomy, 1 (2.0%) high trisomy, may be involved in the upregulation of this gene in sal- 12 (23.5%) low polysomy, and 5 (9.8%) high polysomy ivary duct carcinoma (33, 34). Upregulation of EGFR (Table 2). No correlations between chromosome 17 gene transcription through regulatory proteins and tran- polysomy and immunohistochemical expression, gene scription factors (33) cross-signaling with other growth amplification, age, sex, T and N stage, or clinical outcome factors and via hormonal pathways have been suggested were noted regardless of polysomy categorization (Sup- (34, 35). plementary Table S1). The detection of five tumors with mutations in the EGFR gene in exons 18 and 19 suggests TKIs such as Correlations between HER-2 and EGFR erlotinib may indeed provide targeted therapeutic op- Similar expression profiles of EGFR and HER-2 were tions for a subset of salivary duct carcinoma patients. present in 22 (33.3%) of tumors (5 at 3+, 2 at 2+, 0 at Several of these activating mutations were identified at 1+, and 17 at 0+). Three of the eight HER-2–amplified sites that have previously correlated with high response tumors also had chromosome 7 (EGFR) polysomy (>2.5 rates to TKIs gefitinib or erlotinib in lung adenocarcino- average copy number). Polysomy of chromosomes 7 and mas (21–23). We noted, however, that cases with muta- 17 was found in tumors with different levels of EGFR and tions manifested variable levels of EGFR expression. This HER-2 protein expression (Supplementary Table S2). No finding as previously noted in lung carcinomas suggests positive correlation between EGFR and HER-2 alterations that immunohistochemistry is a poor surrogate for mu- was found (P = 0.008), suggesting independent roles for tational status/clinical response to TKIs (9, 36–38). There- these markers in salivary duct carcinoma. fore, molecular testing of the EGFR gene sequence is recommended to identify the subset of patients likely to Discussion benefit from these targeted TKIs like erlotinib. Moreover, we identified chromosome 7 polysomy in a quarter of Our concurrent analysis, the most comprehensive thus salivary duct carcinomas. Recent studies in lung and far, of the expression and the genomic alterations of the colonic carcinomas have suggested that high polysomy

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for chromosome 7 may also predict responsiveness to Jeahne et al.(8) where the HER-2 high (3+) expression in TKIs perhaps by reducing the rate of resistance to such the 50 salivary duct carcinomas in that cohort was 20.6%, therapies (25, 38). Thus, as more correlative information similar to ours and others (3), although was notably lower becomes available, molecular assessment through FISH than in smaller reports (41, 44), which may reflect group- analysis may provide a predictive assessment for thera- ing of 2+ in the high-expression category and tumor selec- peutic response. tion by immunoexpression in some studies. It is worth Our results also show significant survival differences noting, however, that the incidence of HER-2 expression between patients with and without chromosome 7 poly- was slightly lower in this study (15.2%, 3+) than in our somy (Fig. 2). Although, the rate-limiting polysomy previous report that included a majority of these tumors number of biological significance is not known, our ana- (3). The difference can be attributed to intratumoral hete- lysis of categorical groupings showed that even low levels rogeneity (18, 45) of HER-2 and utilization of microarray of chromosomal duplication affected rate of progression sections, which underscores that certain markers could to death (Table 2, Fig. 3). Similar to other carcinomas, be underestimated using tissue array or on small biopsy polysomy/EGFR copy number in our study correlated specimens. with poor prognosis, suggesting DNA aneuploidy and/ HER-2 gene amplification in a subset of these tumors or relative increase in EGFR copy number plays an highlights the potential therapeutic use of trastuzumab adverse biological role (14, 24, 26–28). Interestingly, pre- (Herceptin) in the management of selected patients simi- vious reports have shown that response to cetuximab and lar to the treatment in mammary carcinoma (46). Trastu- panitumumab, two EGF inhibitors (TKIs), correlated with zumab, a humanized monoclonal antibody, is directed EGFR copy number (polysomy and amplification) in co- against the extracellular domain of the HER-2 tyrosine ki- lonic adenocarcinoma (25) and not mutational status. nase receptor. Identifying the subset of patients who may Similar correlation was noted in lung carcinomas, with benefit from trastuzumab is important to target the pa- response to gefitinib though EGFR mutations and tients who may benefit from treatment (47). This is further HER-2 amplification also frequently present in this supported by initial evidence of response of salivary duct subgroup of patients (31, 39). The low-level amplifi- carcinoma to trastuzumab (14, 48, 49). cation (>2.5 chromosomal copies/cell) found in this In conclusion, our results show that molecular altera- study suggests that even a small increase in gene dose tions of the EGF family of growth receptors, now known may be clinically significant and needs to be targeted to be indicators (possible biomarkers) for potential tar- by a therapeutic agent. geted therapy, are present in a subset of salivary duct car- Heterodimerization of EGFR and HER-2 growth factor cinomas. Both activating mutations (10%) in the EGFR receptors may also cooperate to promote tumor growth gene and chromosome 7 polysomy (25%) were identified and progression and may provide a rationale for the use in a subset of salivary duct carcinomas. These alterations of therapeutic agents or agents with duel targets (34, 40). have correlated with tyrosine kinase inhibitor treatment As 20% of salivary duct carcinomas express combined EGF response in different adenocarcinomas and may potential- and HER-2 receptors, identification of this population may ly be applicable in salivary duct carcinoma patients. Addi- be required for more directed therapy. Moreover, lapatinib tionally, chromosome 7 polysomy portends a poorer ditosylate, a small-molecule inhibitor of both EGFR and clinical outcome that trends with high EGFR expression. HER-2 kinase activity, has shown response in breast adeno- The finding of both overexpression of HER-2 and gene carcinoma, whereas gefitinib, which only blocks EGFR, has amplification defines a group of patients who may benefit not improved response in that patient population (10). from targeted therapy with trastuzumab. The results of this These results suggest that blocking multiple members of study highlight the spectrum of alterations in the EGFR the growth factor receptor pathway may lead to better re- family of receptors as potential targets for therapy in sali- sponse in a subset of these patients. Such complex interac- vary duct carcinomas. tions of the EGFR pathway and targeted agents will require further investigations with careful correlation to uncover Disclosure of Potential Conflicts of Interest the mechanisms leading to therapeutic response. Our results support those of others that HER-2 ampli- No potential conflicts of interest were disclosed. fication is present predominantly in tumors with high (3+) expression (41, 42) and seems to be the dominant mech- Grant Support anism for HER-2 overexpression in this tumor type. This finding suggests that immunohistochemistry evaluation Kenneth D. Müller Professorship, National Cancer Institute Specialized may reflect the functional status of this gene in most sali- Program of Research Excellence (SPORE) grant in Head and Neck Cancer, vary duct carcinomas although amplification is notably and the National Cancer Institute Grant CA-16672. The costs of publication of this article were defrayed in part by the lower (60-80%) than breast adenocarcinomas (90%) with payment of page charges. This article must therefore be hereby marked high (3+) HER-2 expression (7, 41–43). Moreover, the inci- advertisement in accordance with 18 U.S.C. Section 1734 solely to dence of HER-2 expression by immunohistochemistry in indicate this fact. salivary duct carcinomas is similar to breast adenocarci- Received 02/06/2009; revised 12/31/2009; accepted 01/27/2010; noma (15). Our findings are consistent with those of published OnlineFirst 04/06/2010.

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2274 Clin Cancer Res; 16(8) April 15, 2010 Clinical Cancer Research

Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance

Michelle D. Williams, Dianna B. Roberts, Merrill S. Kies, et al.

Clin Cancer Res 2010;16:2266-2274. Published OnlineFirst April 14, 2010.

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