Originally published as JCO Early Release 10.1200/JCO.2007.14.8957 on March 17 2008
Journal of Clinical Oncology, Vol 26, No 11 (April 10), 2008: pp. 1789-1796
© 2008 American Society of Clinical Oncology.
Immunoglobulin G Fragment C Receptor Polymorphisms and Clinical Efficacy of Trastuzumab-Based Therapy in Patients With HER-2/neu–Positive Metastatic Breast Cancer
Antonino Musolino,
Nadia Naldi,
Beatrice Bortesi,
Debora Pezzuolo,
Marzia Capelletti,
Gabriele Missale,
Diletta Laccabue,
Alessandro Zerbini,
Roberta Camisa,
Giancarlo Bisagni,
Tauro Maria Neri,
Andrea Ardizzoni
From the Medical Oncology Unit and Medical Genetics Unit; Laboratory of Viral Immunopathology, Department of Infectious Diseases and Hepatology, University Hospital of Parma, Parma; and the Department of Oncology, S. Maria Nuova Hospital, Reggio Emilia, Italy
Corresponding author: Antonino Musolino, MD, Medical Oncology Unit, University Hospital of Parma, via Gramsci 14, 43100 Parma, Italy; e-mail: antoninomusolino{at}hotmail.com
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ABSTRACT
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Purpose The anti–HER-2/neu monoclonal antibody trastuzumab has been shown to engage both activatory (fragment C receptor [Fc R]IIIa; FcRIIa) and inhibitory (FcRIIb) antibody receptors and FcR polymorphisms have been identified that may affect the antibody-dependent cell-mediated cytotoxicity (ADCC) of natural-killer cells/monocytes. In this study, we tested whether FcR polymorphisms are associated with clinical outcome of patients with breast cancer who received trastuzumab.
Patients and Methods Fifty-four consecutive patients with HER-2/neu–amplified breast cancer receiving trastuzumab plus taxane for metastatic disease were evaluated for genotype for the FcRIIIa-158 valine(V)/phenylalanine(F), FcRIIa-131 histidine(H)/arginine(R), and FcRIIb-232 isoleucine(I)/threonine(T) polymorphisms. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was measured by chromium-51 release using a HER-2/neu–expressing human breast cancer cell line as a target. Controls comprised thirty-four patients treated with taxane alone.
Results Our population was in Hardy-Weinberg equilibrium except for the FcRIIb polymorphism. The FcRIIIa-158 V/V genotype was significantly correlated with objective response rate (ORR) and progression-free survival (PFS). Also, there was trend significance in ORR and PFS for the FcRIIa-131 H/H genotype. The combination of the two favorable genotypes (VV and/or H/H) was independently associated with better ORR and PFS compared with the other combinations. The ADCC analysis showed that V/V and/or H/H PBMCs had a significantly higher trastuzumab-mediated cytotoxicity than PBMCs harboring different genotypes.
Conclusion These data support for the first time the hypothesis that FcR-mediated ADCC plays an important role in the clinical effect of trastuzumab. Prospective studies are needed to confirm the role of FcR polymorphisms in predicting clinical outcome of patients with breast cancer treated with trastuzumab-based therapy.
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INTRODUCTION
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The humanized anti-HER-2/neu immunoglobin G (IgG) 1 monoclonal antibody (mAb), trastuzumab, is an effective treatment for HER-2/neu–positive breast cancer. However, only 25% to 30% of patients with HER-2/neu–positive breast cancers will respond to this mAb and the exact mechanism of its antitumor effect is not clear. Direct antiproliferative and pro-apoptotic effects have been suggested by studies with trastuzumab in vitro on HER-2/neu–positive breast cancer cell lines.1 Immune mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity, may also be involved. This notion has been supported by several observations: trastuzumab had a significantly reduced antitumor effect in IgG fragment C receptor (FcR) -deficient mice2; HER-2/neu–positive breast cancer cell lines are susceptible to ADCC and to complement-mediated cytotoxicity in the presence of trastuzumab3-5; and in vivo acitivity of trastuzumab has been correlated with significantly increased numbers of peritumoral lymphomonocytes and in vitro ADCC.5,6
In ADCC, the antibody binds to tumor cells and then is engaged by effector cells via their receptors for IgG. One group of IgG Fc receptors, FcRs, are expressed on leukocytes and are composed of three distinct classes: FcRI, FcRII, and FcRIII. In humans, the latter two classes can be further divided into FcRIIa and FcRIIb and FcRIIIa and FcRIIIb. The receptors are also distinguished by their affinity for IgG. FcRI exhibits a high affinity for IgG and can bind to monomeric IgG. In contrast, FcRII and FcRIII show a weaker affinity for monomeric and hence can only interact effectively with multimeric immune complexes. Some of these isoforms do not trigger cytotoxicity of human targets—such as FcRIIIb on polymorphonucleates—or initiate inhibitory signals—such as FcRIIb on monocytes/macrophages and polymorphonucleates. However, FcRIIa and FcRIIIa are activating FcRs that are expressed on monocytes/macrophages and on both monocytes/macrophages and natural killer cells, respectively. Although most immune cells coexpress both activatory and inhibitory FcRs, natural killer cells are unique in that they constitutively express only the activating, low-affinity receptor FcRIIIa.7
It is known that the genomic polymorphism (genetic polymorphism) corresponding to the phenotype expression of valine (V) or phenylalanine (F) at amino acid 158 on the FcRIIIa greatly influences the affinity of IgG1 to the Fc receptor.8 More specifically, immune effector cells bearing the FcRIIIa V allele mediate ADCC of anti-HER-2/neu IgG1 variants better than cells bearing the F allele.9
The FcRIIa receptor binds to different classes of IgGs, with a specificity for human (h) IgG1 and hIgG3. Binding to hIgG2 and murine (m) IgG1 depends on an histidine (H)/arginine (R) polymorphism at position 131 of FcRIIa.10 The recent demonstration in follicular lymphoma patients that the response to rituximab (anti-CD20 IgG1 mAb sharing the same FcRs with trastuzumab) is dependent on specific FcRIIa and FcRIIIa polymorphisms supports the importance of ADCC in the in vivo actions of mAbs.11 In that study, patients with homozygous 158 valine/valine (V/V) alleles of FcRIIIa and/or homozygous 131 histidine/histidine (H/H) alleles of FcRIIa showed a higher response rate to rituximab treatment.
The inhibitory FcRIIb has also been shown to play a role in the action of rituximab and trastuzumab in an animal model.2 A recent study reported a polymorphism of isoleucine (I) and threonine (T) at position 232 of FcRIIb involving the transmembrane domain of the protein. The frequency of the 232 T/T genotype was significantly increased in patients with systemic lupus erythematosus compared with healthy individuals, suggesting a potential role for the FcRIIb 232 I/T polymorphism in the balance between activatory and inhibitory FcRs.12
Based on these reports, the objective of this study was to assess the role of FcRIIIa, FcRIIa, and FcRIIb polymorphisms in predicting clinical efficacy of trastuzumab in patients with metastatic HER-2/neu–positive breast cancer.
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PATIENTS AND METHODS
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Patient Population
This study included 54 consecutive patients with HER-2/neu–positive metastatic breast cancer, treated with trastuzumab plus taxane (paclitaxel or docetaxel) as first-line therapy at the Medical Oncology Units of Parma and Reggio Emilia between May 2001 and October 2006. They were selected because of the availability of their tumor and peripheral blood samples, and their evaluable clinical response to trastuzumab-based therapy. The HER-2/neu amplification of all patient cases, as determined by fluorescence in situ hybridization (FISH), was reviewed. As controls, we evaluated 34 consecutive patients with metastatic breast cancer, not selected for HER-2/neu amplification, who received first-line taxane without trastuzumab over the same period. The Response Evaluation Criteria in Solid Tumors were used for disease response assessment.13 FcR polymorphisms were analyzed in all 88 patients (54 study cases and 34 controls). In addition, peripheral blood mononuclear cells (PBMCs) of the patients treated with trastuzumab were drawn at baseline and used for in vitro ADCC assay. This study was conducted according to a protocol approved by the institutional review board/independent ethics committee, and informed consent was obtained from all patients for the use of blood and tissue samples and the analysis of clinical information.
Analysis of FcRIIIa, FcRIIa, and FcRIIb Polymorphisms
Genotyping at the FcR locus is complex in view of the high level of structural homology between the three class II (FcRIIa, FcRIIb and FcRIIc) and two class III (FcRIIIa and FcRIIIb) receptors.14
Total RNA was purified from peripheral-blood mononuclear cells with the Chomczynski and Sacchi method,15 using Total RNA Isolation reagent (Sigma-Aldrich, United Kingdom) and reverse transcribed into cDNA with the GeneAMP RNA PCR Control Kit (Applera, Foster City, CA). Genomic DNA was purified using DNA extraction kit (QIAmp DNA blood mini kit, Qiagen, Valencia, CA).
FcRIIa genotyping, as well as FcRIIIa genotyping, was performed on genomic DNA by polymerase chain reaction (PCR) followed by direct sequencing in both forward and reverse directions, using previously described methods.8,16 FcRIIb analysis was performed on cDNA using a nested PCR approach.12 The resulting PCR product was genotyped by direct sequencing in both forward and reverse direction.
Molecular data were independently interpreted by two biologists (N.N. and B.B.) who were unaware of the clinical outcome of study patients. All PCR conditions are available on request.
ADCC Assay
PBMCs were obtained by Ficoll gradient centrifugation of heparinized blood and were stained with the following antibodies: CD3-FITC (Sigma Chemical Co, St Louis, MO), CD16-PE (PharMingen BD Biosciences, Becton Dickinson, San Jose, CA), CD8-PerCP (Becton Dickinson), and CD56-APC (Pharmigen BD Biosciences).
ADCC was measured in a 4-hour chromium-51 release assay using the HER-2/neu–positive MDA-MB-361 human breast cancer cell line as target and PBMCs as effector cells.5 Cytotoxicity was measured by incubating effector and target cells with trastuzumab, rituximab (control IgG1 mAb), or medium alone. The percentage of cytolysis was calculated according to the formula: cytotoxicity (%): [(experimental counts per minute [cpm] – spontaneous release cpm)/(maximal release cpm – spontaneous release cpm)] x 100. ADCC was calculated according to the formula: ADCC (%) = cytotoxicity (%) – antigen-independent cellular cytotoxicity (%), where antigen-independent cellular cytotoxicity is the nonspecific cytotoxicity in the absence of antibody. To compare different samples, ADCC was normalized by dividing the specific lysis by the frequency of CD16-positive cells calculated on the total PBMCs.
Statistical Analysis
The clinical characteristics and treatment outcomes of the patients were compared according to the FcR polymorphisms, using  2 test17 and two-tailed Fisher's exact test.18 A logistic regression analysis including age (< 60 v  60 years), menopausal status, tumor grade (1 and 2 v 3), Ki67 growth fraction (< 15% v 15%), hormonal receptor status, performance status (Eastern Cooperative Oncology Group 0 v 1), site of metastases (visceral v nonvisceral), taxane administered (paclitaxel v docetaxel), and FcRIIIa, FcRIIa, and FcRIIb genotype was used to identify independent prognostic variables influencing the objective responses. Odds ratio and its 95% CI were also calculated by logistic regression analysis.
Survival estimates were calculated using the Kaplan-Meier method.19 Progression-free survival (PFS) was calculated from the date chemotherapy was started to the earliest date of disease progression or death from breast cancer. Differences in PFS according to the FcR polymorphisms were compared using the log-rank test.20 For the multivariate analysis using Cox's proportional hazard model21 to define independent prognostic factors for PFS, the same variables included for the logistic regression were used. The hazard ratio and 95% CI were also estimated. The statistical data were obtained using SPSS software, version 8.0 (SPSS Inc, Chicago, IL).
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RESULTS
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Patient Characteristics
Clinical and pathologic features of patients treated with trastuzumab and controls are presented in Table 1. Study patients received trastuzumab at a loading dose of 4 mg/kg followed by 2 mg/kg per week until disease progression.
Genotypic Frequencies of Polymorphisms
The FcR genotype frequencies did not differ significantly between study patients and controls (Table A1, online only). They were not significantly different than what would be expected if each group was in Hardy-Weinberg equilibrium, except for the FcRIIb polymorphism in patients treated with trastuzumab (Table A1, online only). When 2-locus linkage disequilibrium analyses were conducted among the three FcR polymorphisms in each study group, no linkage disequilibrium was observed between FcRIIa and FcRIIIa, FcRIIa and FcRIIb, and FcRIIIa and FcRIIb (Table A1, online only).
Clinical Response to Trastuzumab Therapy and FcR Polymorphisms
There was no significant difference in pretreatment features between the V/V, V/F, and F/F 158 FcRIIIa polymorphism groups (Table 2). Similarly, no significant differences between genotype groups were observed when pretreatment patient characteristics were evaluated according to FcRIIa and FcRIIb polymorphisms (Table 2).
A significant difference in objective response rate was observed between patients with FcRIIIa 158 V/V and patients with either 158 V/F or 158 F/F genotype (82% v 42% v 35%; P = .03). Although not statistically significant, patients with FcRIIa 131 H/H also showed a higher response rate than the other two FcRIIa 131 genotypes (70% for H/H v 44% for H/R v 40%; P = .17). There was no significant difference in clinical response to trastuzumab treatment by FcRIIb 232 I/T polymorphism (Table 3).
As shown in previously reported data on patients with lymphoma treated with rituximab,11 the possibility of an association between FcRIIIa and FcRIIa genotypes in predicting clinical response to trastuzumab was examined by analyzing the combination of 158 V/V and/or 131 H/H according to treatment response rate (Table 3). Patients with 158 V/V and/or 131 H/H (total of 17 patients) had a significantly higher response to trastuzumab therapy than patients without either genotype (71% v 38%; P = .04).
By logistic regression analysis, the combination of the two favorable FcRIIIa and FcRIIa genotypes (158 V/V and/or 131 H/H) was identified as the only independent predictive factor for response (odds ratio, 8.7; 95% CI, 1.4 to 53.8; P = .02).
PFS Analysis According to FcR Polymorphisms
The median PFS to trastuzumab-based therapy was 16.8 months. The PFS estimate of patients with FcRIIIa 158 V/V was significantly longer than for patients with 158 V/F, 158 F/F, or F carriers (V/F + F/F combined; Fig 1A). The median PFS was not reached for 158 V/V compared with a median PFS of 15, 11.1, and 12.9 months for each other group, respectively. The median PFS was 29.5 months for patients with FcRIIa 131 H/H, 16.8 for H/R, 10.0 for R/R, and 13.4 for R carriers (H/R + R/R). There was no statistical difference in PFS for patients with 131 H/H compared with patients with other genotypes, except for 131 R/R (P = .04; Fig 1B). In our population, FcRIIb 232 I/T polymorphism did not correlate with PFS to trastuzumab therapy. Median PFS was 15, 22, and 9 months for 232 I/I, I/T, and T/T, respectively (log-rank, P = .98).
When comparing PFS according to the combination of FcRIIIa and FcRIIa genotypes, the PFS estimate for patients with 158 V/V and/or 131 H/H was significantly longer than for patients without either genotype (P = .01), with a median of 30.3 and 12.8 months, respectively (Fig 2). As was observed for logistic regression analysis of response rate, Cox's multivariate analysis showed that the combination of 158 V/V and/or 131 H/H was found to be the only independent predictive factor for PFS (hazard ratio, 5.3; 95% CI, 1.6 to 16.9; P = .005).
Trastuzumab-Mediated ADCC and FcR Polymorphisms
PBMCs of all 54 study cases were used for in vitro ADCC assay. As expected, the control mAb rituximab did not mediate ADCC in HER-2/neu–positive human breast cancer cells (data not shown). When trastuzumab-mediated ADCC was evaluated according to FcR polymorphisms, the FcRIIIa 158 V/V genotype was associated with higher ADCC than 158 V/F and F/F (P = .04; Fig 3A). The 131 H/H genotype also correlated with a better ADCC than 131 H/R and R/R, although without statistical significance (Fig 3B). Similar to that observed for response rate and PFS, ADCC analysis showed that the combination of 158 V/V and/or 131 H/H had a significantly higher trastuzumab-mediated cytotoxicity than other genotypes (P = .04; Fig 3C). Interestingly, in vitro ADCC of PBMCs correlated with in vivo response to trastuzumab (P = .02; Fig 3D). Among the 34 patients treated by taxane without trastuzumab, there was no correlation between FcR polymorphisms and clinical response, and between FcR polymorphisms and PFS (Table A2, online only).
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DISCUSSION
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This study provides evidence in support of the hypothesis that FcR polymorphisms may modulate the clinical efficacy of trastuzumab-based therapy in patients with metastatic HER/2/neu–positive breast cancer. The FcRIIIa 158 V/V genotype, alone and in combination with the FcRIIa H/H genotype (158 V/V and/or 131 H/H), was significantly associated with better response rate and PFS to trastuzumab compared with other FcR genotypes. There was no clinical impact of FcR polymorphisms on patients who received chemotherapy alone.
A few studies tested the hypothesis of an association between clinical activity of therapeutic mAbs and FcRIIIa polymorphism. Three studies showed that FcRIIIa 158 V/V genotype was associated with higher response rate to rituximab treatment in follicular and diffuse large B-cell lymphoma patients.11,22,23 However, this finding may not be applicable to all B-cell malignancies, as suggested by a study of patients with chronic lymphocytic leukemia receiving rituximab, which reported negative results.24 Other mAbs were analyzed according to the FcRIIIa polymorphism. An association was observed between FcRIIIa 158 V/V and decrease in C-reactive protein after infliximab (recombinant anti-tumor necrosis factor  IgG1 mAb) in Crohn's disease.25 FcRIIIa polymorphism did not predict response to alemtuzumab (humanized anti-CD52 IgG1 mAb) in patients with chronic lymphocytic leukemia.26 Interestingly, a study identified the predictive value of FcRIIIa 158 V/V on clinical outcome in patients with lymphoma who received active immunotherapy with idiotype vaccination.27 This result implies that the antibodies induced against a tumor antigen are clinically relevant by killing antibody-coated tumor cells through FcR-bearing lymphomonocytes.
In our study, as previously reported in patients with lymphoma treated with rituximab and in patients with metastatic colorectal cancer treated with cetuximab (chimeric anti-EGFR IgG1 mAb),11,28 a trend toward better response rate and PFS to trastuzumab was observed for the FcRIIa 131 H/H genotype. In multivariate analysis, the combination of FcRIIIa 158 V/V and/or FcRIIa 131 H/H was found to be the only independent predictive factor for clinical outcome to trastuzumab therapy. These findings were not a result of a linkage disequilibrium between FcRIIa and FcRIIIa polymorphisms. There is in fact a random distribution of combinations of variant genotypes of FcRIIa and FcRIIIa in the normal population.29 In contrast to data reported for hIgG2,10,30 a different affinity of the two FcRIIa allelic forms for hIgG1 has not yet been clearly demonstrated.10 The biologic explanation for the association observed between FcRIIa polymorphism and trastuzumab activity remains, therefore, to be elucidated. The possibility that other unidentified polymorphic genes are linked to the 131 H/R locus and are responsible for the association with clinical outcome to trastuzumab, can not be excluded.
Consistent with previous reports on patients treated with rituximab and idiotype vaccine,11,27 our study also showed that patients with FcRIIIa 158 V/F had a similar outcome to patients with 158 F/F. Similar findings were observed between FcRIIa 131 H/R heterozygous and 131 R/R homozygous patients. The biologic explanation of this phenomenon is unclear, because heterozygous patients would be expected to have an intermediate clinical course. It is, however, possible that a certain threshold for FcR-delivered signal is required to activate the effector cells fully, which can only be reached by signaling through the product of two V, or H, alleles.
We showed no relationship between FcRIIb 232 I/T polymorphism and clinical outcome to trastuzumab therapy. However, the FcRIIb genotype frequencies observed were not in Hardy-Weinberg equilibrium, and further genotyping analyses of more instances are warranted to confirm this negative result.
To confirm the role of ADCC in trastuzumab function, and the role of FcR polymorphisms in mediating ADCC, we tested in vitro the antitumoral activity of mononuclear cells of study patients. As was observed for response rate and PFS, ADCC analysis showed that 158 V/V and/or 131 H/H PBMCs had a significantly higher trastuzumab-mediated cytotoxicity than PBMCs harboring other genotypes. However, the fact that trastuzumab does retain approximately 40% of its antitumor activity in FcR–/– mice compared with wild-type mice,2 indicates that some biologic effects of mAbs can be independent of FcRs. Trastuzumab mechanisms of action other than ADCC can operate in vivo such as complement-dependent cytotoxicity, complement-dependent cell-mediated cytotoxicity,31,32 and/or apoptosis.1,33 ADCC could therefore be viewed as an additional mechanism in the response to trastuzumab that is particularly effective in patients who are FcR 158 V and/or FcRIIa 131 H homozygous.
In conclusion, to our knowledge, this study supports for the first time the hypothesis that FcR polymorphisms play a role in trastuzumab-mediated ADCC and predict clinical outcome of patients with breast cancer treated with trastuzumab-based therapy. Given the retrospective design of our study, further investigation of common variants of FcRIIIa, FcRIIa, and FcRIIb are warranted in a prospectively accrued population.
One of the implications of our results is the possibility to develop mAbs tailored to each patient FcR genotype. Many of the IgG1 residues that are involved in binding to FcRs are different.9 As a consequence, it is possible to generate IgG1 variants with altered FcR-binding characteristics (improved or debilitated).34 Indeed, by modifying various residues in the IgG1 lower hinge region, Shields et al obtained IgG1 mutants that bind more strongly to FcRIIIa 158 F than native IgG1.9,35 Therapeutic IgG1 mAbs with variant Fc portions that improve binding to FcRIIIa 158 F, at least to the level seen for FcRIIIa 158 V, could provide increased therapeutic efficacy to the majority of the population.
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AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
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The author(s) indicated no potential conflicts of interest.
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AUTHOR CONTRIBUTIONS
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Conception and design: Antonino Musolino, Nadia Naldi, Gabriele Missale
Administrative support: Roberta Camisa
Provision of study materials or patients: Antonino Musolino, Nadia Naldi, Beatrice Bortesi, Debora Pezzuolo, Marzia Capelletti, Gabriele Missale, Diletta Laccabue, Alessandro Zerbini, Giancarlo Bisagni
Collection and assembly of data: Antonino Musolino, Nadia Naldi, Beatrice Bortesi, Debora Pezzuolo, Marzia Capelletti, Gabriele Missale, Diletta Laccabue, Alessandro Zerbini, Roberta Camisa, Giancarlo Bisagni
Data analysis and interpretation: Antonino Musolino, Nadia Naldi, Beatrice Bortesi, Debora Pezzuolo, Marzia Capelletti, Gabriele Missale, Diletta Laccabue, Alessandro Zerbini, Roberta Camisa, Tauro Maria Neri, Andrea Ardizzoni
Manuscript writing: Antonino Musolino, Gabriele Missale
Final approval of manuscript: Antonino Musolino, Andrea Ardizzoni
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Appendix
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Glossary Terms
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- Fc Receptors:
- Receptors for immunoglobulin G. They are expressed on leukocytes and are composed of three distinct classes: FcRI, FcRII, and FcRIII. In humans, the latter two classes can be further divided into FcRIIa and FcRIIb and FcRIIIa and FcRIIIb.
- ADCC (antibody-dependent cell-mediated cytotoxicity):
- a mechanism of cell-mediated immunity whereby an effector cell of the immune system actively lyses a target cell that has been bound by specific antibodies.
- Allele:
- an alternative form of a gene (in diploids, one member of a pair) that is located at a specific position on a specific chromosome.
- Linkage disequilibrium:
- the non-random association of alleles at two or more loci, not necessarily on the same chromosome.
- cDNA (complementary DNA):
- DNA synthesized from a mature mRNA template in a reaction catalysed by the enzyme reverse transcriptase.
- Genetic polymorphisms:
- A genetic variant seen in at least 1% of the population. Because proteins are gene products, their polymorphisms reflect allelic differences in the gene. The advent of restriction enzymes, which digest DNA to fragments based on sequence specificity, has ushered in an era of restriction fragment length polymorphisms (RFLPs) in which changes in DNA sequence(s) are manifest as restriction fragments of different size(s) when cleaved with a specific restriction enzyme. Polymorphisms are used in tissue typing, in determining disease, in pharmacogenetics, and in assessing genetic diversity.
- Immunoglobin:
- A class of proteins produced in lymph tissue.
- HER-2/neu (human epithelial growth factor receptor-2):
- Also called ErbB2, HER-2/neu belongs to the EGFR family and is overexpressed in several solid tumors. Like EGFR, it is a tyrosine kinase receptor whose activation leads to proliferative signals within the cells. On activation, the HER family of receptors are known to form homodimers and heterodimers, each with a distinct signaling activity. Because HER-2 is the preferred dimerization partner when heterodimers are formed, it is important for signaling through ligands specific for any members of the family. It is typically overexpressed in several epithelial tumors.
- Genotype:
- The specific genetic makeup of a given individual. Although genotypes give rise to the phenotype of an individual, genotypes and phenotypes are not always correlative. For example, some genotypes are expressed only under specific environmental conditions.
- FISH (fluorescence in situ hybridization):
- In situ hydridization is a sensitive method that is generally used to detect specific gene sequences in tissue sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest. FISH uses a fluorescence probe to increase the sensitivity of in situ hybridization.
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ACKNOWLEDGMENTS
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We thank Paolo Bruzzi, MD, Unit of Epidemiology and Biostatistics, Department of Epidemiology and Prevention, National Cancer Research Institute, Genova, Italy, for external statistical review of the manuscript; and Manlio Ferrarini, MD, Director, Division of Medical Oncology C, IST, Genova, Italy, for his helpful comments and advices regarding this work. The human breast cancer cell line MDA-MB-361 was kindly provided by Francesco Fagnoni, MD, Laboratory of Experimental Oncology, Fondazione S. Maugeri, Pavia, Italy.
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NOTES
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published online ahead of print at www.jco.org on March 17, 2008.
Supported in part by the Cariparma Foundation (Fondazione Cassa di Risparmio di Parma, Parma, Italy).
Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Submitted October 15, 2007;
accepted December 17, 2007.
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ABSTRACT
INTRODUCTION
