PTEN, PIK3CA, p-AKT, and p-p70S6K Status
2010; Elsevier BV; Volume: 177; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090885
ISSN1525-2191
AutoresFrancisco J. Esteva, Hua Guo, Siyuan Zhang, Cesar A. Santa‐Maria, Steven Stone, Jerry S. Lanchbury, Ayşegül A. Şahin, Gabriel N. Hortobágyi, Dihua Yu,
Tópico(s)HER2/EGFR in Cancer Research
ResumoPhosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies. Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% to 25% of invasive breast cancers. Patients with HER2-overexpressing tumors experience a shorter time to relapse and shorter overall survival than patients with tumors of normal HER2 levels.1Slamon DJ Clark GM Wong SG Levin WJ Ullrich A McGuire WL Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235: 177-182Crossref PubMed Scopus (9936) Google Scholar, 2Rohan TE Hartwick W Miller AB Kandel RA Immunohistochemical detection of c-erbB-2 and p53 in benign breast disease and breast cancer risk.J Natl Cancer Inst. 1998; 90: 1262-1269Crossref PubMed Scopus (103) Google Scholar HER2 overexpression can lead to activation of many downstream signaling molecules, including Ras-Gap, Src, phosphoinositide 3-kinase (PI3K)/AKT, and many other signaling events.3Hynes NE MacDonald G ErbB receptors and signaling pathways in cancer.Curr Opin Cell Biol. 2009; 21: 177-184Crossref PubMed Scopus (732) Google Scholar, 4Kurokawa H Arteaga CL ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms.Clin Cancer Res. 2003; 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6: 117-127Abstract Full Text Full Text PDF PubMed Scopus (1535) Google Scholar, 13Metro G Mottolese M Fabi A HER-2-positive metastatic breast cancer: trastuzumab and beyond.Expert Opin Pharmacother. 2008; 9: 2583-2601Crossref PubMed Scopus (41) Google Scholar Despite the large amounts of preclinical and clinical data, the causes of trastuzumab resistance are still poorly understood.14Nahta R Yu D Hung MC Hortobagyi GN Esteva FJ Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer.Nat Clin Pract Oncol. 2006; 3: 269-280Crossref PubMed Scopus (721) Google Scholar The PI3K pathway, downstream of HER2, plays a central role in regulating a number of cellular processes, such as apoptosis, migration, angiogenesis, cell proliferation, and glucose metabolism, and it is involved in trastuzumab resistance.15Esteva FJ Yu D Hung MC Hortobagyi GN Molecular predictors of response to trastuzumab and lapatinib in breast cancer.Nat Rev Clin Oncol. 2010; 7: 98-107Crossref PubMed Scopus (142) Google Scholar, 16Mills GB Kohn E Lu Y Eder A Fang X Wang H Bast RC Gray J Jaffe R Hortobagyi G Linking molecular diagnostics to molecular therapeutics: targeting the PI3K pathway in breast cancer.Semin Oncol. 2003; 30: 93-104Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar PI3K phosphorylates phosphatidylinositols on the cell membrane, generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2). Then, at the cell membrane, PIP3 recruits protein kinases and activates protein kinase B (PKB)/AKT.17Vivanco I Sawyers CL The phosphatidylinositol 3-Kinase AKT pathway in human cancer.Nat Rev Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5137) Google Scholar In breast cancer cells, HER2 overexpression can lead to activation of the PI3K/AKT pathway.18Hynes NE Dey JH PI3K inhibition overcomes trastuzumab resistance: blockade of ErbB2/ErbB3 is not always enough.Cancer Cell. 2009; 15: 353-355Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The activation of AKT and its downstream signaling have been demonstrated to inhibit cell cycle arrest and block trastuzumab-mediated apoptosis.12Yakes FM Chinratanalab W Ritter CA King W Seelig S Arteaga CL Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action.Cancer Res. 2002; 62: 4132-4141PubMed Google Scholar AKT phosphorylation and AKT kinase activities were found to be increased in trastuzumab-resistant cells, derived from BT474 HER2-overexpressing breast cancer cells, when compared with parental cells.19Chan CT Metz MZ Kane SE Differential sensitivities of trastuzumab (Herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors.Breast Cancer Res Treat. 2005; 91: 187-201Crossref PubMed Scopus (78) Google Scholar These data provide insight into the trastuzumab-resistance mechanism of PI3K/AKT signaling.15Esteva FJ Yu D Hung MC Hortobagyi GN Molecular predictors of response to trastuzumab and lapatinib in breast cancer.Nat Rev Clin Oncol. 2010; 7: 98-107Crossref PubMed Scopus (142) Google Scholar Aberrations in the components of the PI3K pathway have been reported in most solid tumors, including breast cancer.16Mills GB Kohn E Lu Y Eder A Fang X Wang H Bast RC Gray J Jaffe R Hortobagyi G Linking molecular diagnostics to molecular therapeutics: targeting the PI3K pathway in breast cancer.Semin Oncol. 2003; 30: 93-104Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar PTEN is a tumor suppressor that dephosphorylates the D3 position of PIP3 and inhibits the PI3K/AKT pathway.20Vazquez F Sellers WR The PTEN tumor suppressor protein: an antagonist of phosphoinositide 3-kinase signaling.Biochim Biophys Acta. 2000; 1470: M21-M35PubMed Google Scholar Loss of PTEN function as a result of mutation, deletion, or promoter methylation has been reported in nearly 50% of breast cancers.11Nagata Y Lan KH Zhou X Tan M Esteva FJ Sahin AA Klos KS Li P Monia BP Nguyen NT Hortobagyi GN Hung MC Yu D PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.Cancer Cell. 2004; 6: 117-127Abstract Full Text Full Text PDF PubMed Scopus (1535) Google Scholar In addition, the gene encoding one of the PI3K catalytic subunits, p110α (PIK3CA), has been found to be mutated in about 25% of breast cancers.21Bachman KE Argani P Samuels Y Silliman N Ptak J Szabo S Konishi H Karakas B Blair BG Lin C Peters BA Velculescu VE Park BH The PIK3CA gene is mutated with high frequency in human breast cancers.Cancer Biol Ther. 2004; 3: 772-775Crossref PubMed Scopus (555) Google Scholar, 22Saal LH Holm K Maurer M Memeo L Su T Wang X Yu JS Malmstrom PO Mansukhani M Enoksson J Hibshoosh H Borg A Parsons R PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma.Cancer Res. 2005; 65: 2554-2559Crossref PubMed Scopus (733) Google Scholar, 23Perez-Tenorio G Alkhori L Olsson B Waltersson MA Nordenskjold B Rutqvist LE Skoog L Stal O PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer.Clin Cancer Res. 2007; 13: 3577-3584Crossref PubMed Scopus (241) Google Scholar Most of the reported mutations are localized to hotspots in exons 9 and 20 of the PIK3CA gene, which result in increased PI3K pathway signaling.22Saal LH Holm K Maurer M Memeo L Su T Wang X Yu JS Malmstrom PO Mansukhani M Enoksson J Hibshoosh H Borg A Parsons R PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma.Cancer Res. 2005; 65: 2554-2559Crossref PubMed Scopus (733) Google Scholar, 24Isakoff SJ Engelman JA Irie HY Luo J Brachmann SM Pearline RV Cantley LC Brugge JS Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells.Cancer Res. 2005; 65: 10992-11000Crossref PubMed Scopus (414) Google Scholar We previously discovered that PTEN activation is a novel mechanism of trastuzumab antitumor function, and PTEN loss confers trastuzumab resistance in HER2-overexpressing breast cancer cells.11Nagata Y Lan KH Zhou X Tan M Esteva FJ Sahin AA Klos KS Li P Monia BP Nguyen NT Hortobagyi GN Hung MC Yu D PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.Cancer Cell. 2004; 6: 117-127Abstract Full Text Full Text PDF PubMed Scopus (1535) Google Scholar PTEN loss significantly predicted poor response to trastuzumab-based therapy in a small cohort of HER2-positive patients with MBC.11Nagata Y Lan KH Zhou X Tan M Esteva FJ Sahin AA Klos KS Li P Monia BP Nguyen NT Hortobagyi GN Hung MC Yu D PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.Cancer Cell. 2004; 6: 117-127Abstract Full Text Full Text PDF PubMed Scopus (1535) Google Scholar Later, it was reported that both low PTEN levels and PI3K-activating PIK3CA mutations contribute to trastuzumab resistance in HER2-overexpressing breast cancer.25Berns K Horlings HM Hennessy BT Madiredjo M Hijmans EM Beelen K Linn SC Gonzalez-Angulo AM Stemke-Hale K Hauptmann M Beijersbergen RL Mills GB van de Vijver MJ Bernards R A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.Cancer Cell. 2007; 12: 395-402Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar, 26Junttila TT Akita RW Parsons K Fields C Lewis Phillips GD Friedman LS Sampath D Sliwkowski MX Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941.Cancer Cell. 2009; 15: 429-440Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar PTEN loss or PIK3CA mutations, which indicate activation of the PI3K pathway, are considered as markers for poor response to trastuzumab in patients with HER2-overexpressing breast cancer.25Berns K Horlings HM Hennessy BT Madiredjo M Hijmans EM Beelen K Linn SC Gonzalez-Angulo AM Stemke-Hale K Hauptmann M Beijersbergen RL Mills GB van de Vijver MJ Bernards R A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.Cancer Cell. 2007; 12: 395-402Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar On the other hand, some studies found no correlation between PTEN expression and trastuzumab response or survival in patients with HER2-positive breast cancer.27Yonemori K Tsuta K Shimizu C Hatanaka Y Hashizume K Ono M Kouno T Ando M Tamura K Katsumata N Hasegawa T Kinoshita T Fujiwara Y Immunohistochemical expression of PTEN and phosphorylated Akt are not correlated with clinical outcome in breast cancer patients treated with trastuzumab-containing neo-adjuvant chemotherapy.Med Oncol. 2008; 26: 344-349Crossref PubMed Scopus (31) Google Scholar, 28Gori S Sidoni A Colozza M Ferri I Mameli MG Fenocchio D Stocchi L Foglietta J Ludovini V Minenza E De Angelis V Crino L EGFR, pMAPK, pAkt and PTEN status by immunohistochemistry: correlation with clinical outcome in HER2-positive metastatic breast cancer patients treated with trastuzumab.Ann Oncol. 2009; 20: 648-654Crossref PubMed Scopus (67) Google Scholar These contradictory findings prompted us to further investigate the association between PTEN status and clinical outcomes in a large cohort of patients with MBC who were treated with trastuzumab-based therapy. We tested the hypothesis that a comprehensive assessment of PI3K pathway activation status provides biomarkers that can identify patients who may not benefit from trastuzumab-based therapy. HER2-positive tumor samples were obtained from 137 eligible patients at the University of Texas M. D. Anderson Cancer Center under an institutional review board–approved protocol. All of the patients, who subsequently developed MBC, were treated with trastuzumab-based therapy between 1998 and 2006. Tumors that scored 3+ on immunohistochemical (IHC) analysis of HER2 and/or had an amplified HER2 gene detected by fluorescence in situ hybridization (FISH) were defined as HER2 positive. PTEN, p-AKT, and p-p70S6K status, determined by IHC analysis, were evaluated on serial slides and scored on all tissue samples, except for one sample on which p-AKT scoring could not be obtained. IHC staining was performed on 4-μm slices of formalin-fixed paraffin-embedded (FFPE) tissue sections with primary antibodies against PTEN (138G6, 1:50; Cell Signaling Technology, Danvers, MA), p-AKT (Ser473, 736E11, IHC specific, Cell Signaling; 1:50), and p-p70S6K (Thr389, 1A5, Cell Signaling, 1:700). To validate the specificity of PTEN antibodies, Western blotting was performed first using both BT474 control cells and BT474 PTEN-deficient cell lines (by shRNA knockdown). Then mouse xenograft tumors derived from above cell lines were embedded in paraffin and used as negative and positive control for IHC staining. After comparing four PTEN antibodies from different companies (Cell Signaling; R&D Systems, Minneapolis, MN; Cascade Bioscience, Winchester, MA; and Dako, Carpinteria, CA), we found that the antibody from Cell Signaling is the most specific in our studies (data not shown). The p-AKT and p-p70S6K stainings were validated by Western blotting and other studies, as previously described.29Klos KS Wyszomierski SL Sun M Tan M Zhou X Li P Yang W Yin G Hittelman WN Yu D ErbB2 increases vascular endothelial growth factor protein synthesis via activation of mammalian target of rapamycin/p70S6K leading to increased angiogenesis and spontaneous metastasis of human breast cancer cells.Cancer Res. 2006; 66: 2028-2037Crossref PubMed Scopus (172) Google Scholar, 30Zhou Y Pan Y Zhang S Shi X Ning T Ke Y Increased phosphorylation of p70 S6 kinase is associated with HPV16 infection in cervical cancer and esophageal cancer.Br J Cancer. 2007; 97: 218-222Crossref PubMed Scopus (34) Google Scholar After deparaffinization and rehydration, slides were subjected to heat-induced epitope retrieval in 10 mmol/L citrate buffer (pH 6.0) for p-p70S6K staining or 1 mmol/L EDTA (pH 8.0) for PTEN and p-AKT staining in a hot water bath (95°C) for 20 minutes. Endogenous peroxidase activity was blocked for 15 minutes in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 hour at room temperature, the sections were incubated with primary antibodies for at least 18 hours at 4°C overnight. Immunodetection was performed using the LSAB2 kit (Dako), 3-3′-diaminobenzidine was used for color development, and hematoxylin was used for counterstaining. Positive control slides (from BT474 xenograft tumors) and negative control slides (without primary antibody) were included within each batch for staining. The PTEN, p-AKT, and p-p70S6K expression levels were evaluated based on the staining intensity (SI) and percentage of positive cells (PP) within the whole tissue section. PTEN was scored semiquantitatively using the immunoreactive score (IRS), which was calculated as follows: IRS = SI × PP. SI was defined as 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. PP was scored as 0 = 1%; 1 = 1%−10%; 2 = 11%−50%; 3 = 51%−80%; and 4 = >80% positive cells. PTEN loss was defined as an IRS of 3 or less.11Nagata Y Lan KH Zhou X Tan M Esteva FJ Sahin AA Klos KS Li P Monia BP Nguyen NT Hortobagyi GN Hung MC Yu D PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.Cancer Cell. 2004; 6: 117-127Abstract Full Text Full Text PDF PubMed Scopus (1535) Google Scholar The p-AKT and p-p70S6K stainings were evaluated by an H score,31Shousha S Oestrogen receptor status of breast carcinoma: allred/H score conversion table.Histopathology. 2008; 53: 346-347Crossref PubMed Scopus (51) Google Scholar which was calculated by multiplying the PP by the corresponding SI (1 = weak, 2 = moderate, and 3 = strong), giving a maximum score of 300 (100% × 3). H scores >50 were considered positive. We isolated genomic DNA from FFPE tumor samples for PIK3CA mutation screening from 90 patients. The other 47 tumors are very small in size and did not allow analysis of PIK3CA mutations. PIK3CA mutation screening was performed by using the QIAamp DNA FFPE tissue kit (Qiagen, Valencia, CA) according to the kit protocol. First, the FFPE slides were stained with hematoxylin and eosin and checked by a pathologist to identify the tumor region. After deparaffinization, most of the tumor was cut out from the slides by a razor blade. For a few samples, dissection was aided by laser capture microscopy owing to the dispersion of the tumor cells. For laser capture, we used a PALM MicroBeam Microscope for Laser Micromanipulation (Carl Zeiss, Inc., Thornwood, NY) with HV-D30 Hitachi camera capture system (Tokyo, Japan). The microscope contains built-in class IIIB and class IV lasers. The equipment was run using PALM Robo Software 4.0.0.10 (Carl Zeiss), and all laser capture was directed by a pathologist. The isolated genomic DNA was diluted to 3 ng/μl with TE buffer (10 mmol/L Tris-HCl, 0.1 mmol/L EDTA [pH 8.0]) and sent to a DNA sequencing facility for sequencing of exons 9 and 20 of PIK3CA. For each sample, we submitted 6 ng of genomic DNA. The DNA was amplified using the primer pair 5′-GTTTTCCCAGTCACGACGTATTGAAAATGTATTTGCTTTTTC and 5′-GGAAACAGCTATGACCATAACATGCTGAGATCAGCCAAAT for exon 9 and the primer pair 5′-GTTTTCCCAGTCACGACGATTAACATCATTTGCTCCAAAC and 5′-AGGAAACAGCTATGACCATGCTGTTTAATTGTGTGGAAGAT for exon 20. The primers also contained M13 forward and reverse tails to facilitate sequencing. After amplification, the DNA sequence was determined on a Mega BASE 4500 (GE Health care, Waukesha, WI) using dye-primer chemistry, as previously described.32Frank TS Deffenbaugh AM Reid JE Hulick M Ward BE Lingenfelter B Gumpper KL Scholl T Tavtigian SV Pruss DR Critchfield GC Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: analysis of 10,000 individuals.J Clin Oncol. 2002; 20: 1480-1490Crossref PubMed Scopus (758) Google Scholar The observed mutations were confirmed by two independent amplification and sequencing reactions. The success rate was 60% (55/90). Response to trastuzumab-based therapy was evaluated every 8 to 12 weeks using the response evaluation criteria in solid tumors (RECIST) with modifications.33Jaffe CC Measures of response: rECIST. WHO, and new alternatives.J Clin Oncol. 2006; 24: 3245-3251Crossref PubMed Scopus (256) Google Scholar A complete response (CR) was defined as the complete disappearance of all radiographically or visually apparent tumors for at least 4 weeks. A partial response (PR) was defined as a ≥30% decrease in the maximum diameter of measurable metastatic lesions for at least 4 weeks, with no new lesions appearing. Stable disease (SD) was defined as no change in the size of measurable lesions ≥20% and no appearance of new lesions. Progressive disease (PD) was defined as the appearance of any new lesion or an increase in the size of the existing lesions ≥20%. In this study, response was defined as a CR, PR, or SD longer than 6 months (clinical benefit = CR + PR + SD). Overall survival (OS) was defined as the time from initiation of trastuzumab treatment to the patient's death or the final day of follow-up. The relationship between the expression of the biomarkers and response to trastuzumab and clinicopathologic features was analyzed using Fisher's exact test and the χ2 test. Survival analyses were performed using Kaplan–Meier curves with log-rank tests. A P value <0.05 (two-sided) was considered to be statistically significant. All statistical analyses were performed using the SPSS 16.0 program (SPSS, Chicago, IL). The study population comprised 137 patients with HER2-positive MBC who received trastuzumab treatment. The patients' clinical characteristics are summarized in Table 1. The median age at first diagnosis was 44 years (range, 20–73 years). Of the 137 patients, 123 (89.8%) were initially diagnosed with invasive ductal carcinoma. Most patients were white (104, 75.9%); 54 patients (39.4%) had estrogen receptor (ER)-positive tumors, and 44 (32.1%) had progesterone receptor (PR)-positive tumors. The median disease-free survival time before developing metastatic disease was 18 months (range, 0–169 months), and most patients (51.8%) had multiple distant metastases. Among the 137 patients, the three most frequent first metastatic sites were the liver (65, 47.4%), bone (60, 43.8%), and lungs (52, 38.0%). During follow-up, 45 patients (32.8%) developed brain metastases, of which 31 (31/45 = 68.9%) were diagnosed with brain metastasis after initiation of Trastuzumab-based therapy.Table 1Clinicopathological Characteristics of 137 Metastatic Breast Cancer Patients Treated with Trastuzumab-Based TherapyCharacteristicn (%)Age at diagnosis (median 44 years, range 20–73) <50 years89 (65) ≥50 years48 (35)Nuclear grade I0 II22 (16) III106 (77.4) Unknown9 (6.6)Histologic type Invasive ductal carcinoma123 (89.8) Invasive mixed ducal/lobular carcinoma8 (5.8) Invasive lobular carcinoma2 (1.5) Inflammatory carcinoma1 (0.7) Invasive micropapillary carcinoma1 (0.7) Breast cancer, NOS2 (1.5)Race/ethnicity White104 (75.9) Black8 (5.8) Hispanic17 (12.4) Asian6 (4.4) Others2 (1.5)ER status Positive56 (40.9) Negative78 (56.9) Unknown3 (2.2)PR status Positive45 (32.8) Negative85 (62.0) Unknown7 (5.1)First site of metastasis Liver65 (47.4) Bone60 (43.8) Lung52 (38) Brain9 (6.6)Brain metastasis Total cases from time of original diagnosis45 (32.8)ER indicates estrogen receptor; PR, progesterone receptor. Open table in a new tab ER indicates estrogen receptor; PR, progesterone receptor. The patients' demographic characteristics had no relationship with response to trastuzumab or overall survival after trastuzumab treatment, except for race. Black patients had the shortest OS after starting trastuzumab (P = 0.014) compared with other groups. Information on the patients' trastuzumab-based therapy and patients' clinical outcomes were summarized in Table 2. Of the 137 patients, 87 (63.5%) used trastuzumab treatment as first-line therapy, and 91 (66.4%) had received no prior chemotherapy. Eighty-two patients (59.9%) responded to trastuzumab-based therapy (CR + PR + SD), with a median OS of 30 months (range, 2–84 months). However, 55 patients (40.1%) were resistant to trastuzumab (PD), with a median OS of only 13 months (range, 1–49, months; P < 0.001 compared with CR + PR + SD group).Table 2Trastuzumab-Based Therapy Information and Clinical Outcomes of the 137 Metastatic Breast Cancer PatientsVariableDescriptionTtzm-based therapy Ttzm single agent16 (11.7%) Ttzm + navelbine30 (21.9%) Ttzm + taxane-based chemotherapy76 (55.5%) Ttzm + other therapy15 (10.9%)Lines of Ttzm-based therapy First-line87 (63.5%) Second-line26 (19.0%) Other24 (17.5%)Prior chemotherapy Without91 (66.4%) With39 (28.5%) Unknown7 (5.1%)Response to Ttzm-based therapy CR + PR + SD82 (59.9%) PD55 (40.1%)Outcome Dead97 (70.8%) Alive40 (29.2%)Median time of overall survival from Ttzm-based therapy start date23m (range 1–84 m) Patients responsive (CR + PR + SD) to therapy30m (range 2–84 m) Patients resistant (PD) to therapy13m (range 1–49 m)Ttmz indicates trastuzumab; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. 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