title: Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.
abstract: BACKGROUND
  Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab.
  
  
  METHODS
  Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab.
  
  
  RESULTS
  We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006).
  
  
  CONCLUSIONS
  Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.
authors.affiliation: Unit of Molecular Oncology and Angiogenesis, IRCCS AOU San Martino-IST, Genoa, Italy. silviaboero@hotmail.com.
authors.forename: Silvia
authors.initials: S
authors.lastname: Boero
authors.affiliation: Unit of Tumor Epigenetics, IRCCS AOU San Martino-IST, Genoa, Italy. anna.morabito@hsanmartino.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Morabito
authors.affiliation: Unit of Tumor Epigenetics, IRCCS AOU San Martino-IST, Genoa, Italy. barbara.banelli@hsanmartino.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Banelli
authors.affiliation: Development of Innovative Therapies Unit, IRCCS AOU San Martino-IST, Genoa, Italy. barbara.cardinali@hsanmartino.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cardinali
authors.affiliation: Clinical Epidemiology Unit, IRCCS AOU San Martino-IST, Genoa, Italy. beatrice.dozin@hsanmartino.it.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Dozin
authors.affiliation: Medical Oncology Unit, Sacro Cuore Don Calabria Hospital, Negrar, Verona, Italy. gianluigi.lunardi@sacrocuore.it.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Lunardi
authors.affiliation: Cellular Biology Unit, IRCCS AOU San Martino-IST, Genoa, Italy. patrizia.piccioli@hsanmartino.it.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Piccioli
authors.affiliation: Laboratory of Molecular Diagnostics, IRCCS AOU San Martino-IST, Genoa, Italy. sonia.lastraioli@hsanmartino.it.
authors.forename: Sonia
authors.initials: S
authors.lastname: Lastraioli
authors.affiliation: Unit of Tumor Epigenetics, IRCCS AOU San Martino-IST, Genoa, Italy. roberta.carosio@yahoo.it.
authors.forename: Roberta
authors.initials: R
authors.lastname: Carosio
authors.affiliation: Unit of Pathology, IRCCS AOU San Martino-IST, Genoa, Italy. sandra.salvi@hsanmartino.it.
authors.forename: Sandra
authors.initials: S
authors.lastname: Salvi
authors.affiliation: Development of Innovative Therapies Unit, IRCCS AOU San Martino-IST, Genoa, Italy. alessia.levaggi@hsanmartino.it.
authors.forename: Alessia
authors.initials: A
authors.lastname: Levaggi
authors.affiliation: Unit of Medical Oncology 2, IRCCS AOU San Martino-IST, Genoa, Italy. francesca.poggio.1987@gmail.com.
authors.forename: Francesca
authors.initials: F
authors.lastname: Poggio
authors.affiliation: Development of Innovative Therapies Unit, IRCCS AOU San Martino-IST, Genoa, Italy. alessia.dalonzo@hsanmartino.it.
authors.forename: Alessia
authors.initials: A
authors.lastname: D'Alonzo
authors.affiliation: Unit of Tumor Epigenetics, IRCCS AOU San Martino-IST, Genoa, Italy. massimo.romani@hsanmartino.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Romani
authors.affiliation: Development of Innovative Therapies Unit, IRCCS AOU San Martino-IST, Genoa, Italy. lucia.delmastro@hsanmartino.it.
authors.forename: Lucia
authors.initials: L
authors.lastname: Del Mastro
authors.affiliation: Unit of Molecular Oncology and Angiogenesis, IRCCS AOU San Martino-IST, Genoa, Italy. alessandro.poggi@hsanmartino.it.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Poggi
authors.affiliation: Unit of Tumor Epigenetics, IRCCS AOU San Martino-IST, Genoa, Italy. mariapia.pistillo@hsanmartino.it.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Pistillo
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D015331:Cohort Studies; D005260:Female; D005434:Flow Cytometry; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D007150:Immunohistochemistry; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000166:cytology; D061986:MCF-7 Cells; D008875:Middle Aged; D009362:Neoplasm Metastasis; D020641:Polymorphism, Single Nucleotide; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D017452:Receptors, IgG / Q000235:genetics*; D017422:Sequence Analysis, DNA; D000068878:Trastuzumab / Q000627:therapeutic use*
chemical_list: D000970:Antineoplastic Agents; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG; D000068878:Trastuzumab; C496124:FCGR3A protein, human; C080568:Fc gamma receptor IIA; C508053:ERBB2 protein, human