Purpose: We investigated the antitumor activity of the combination of two different humanized monoclonal human epidermal growth factor receptor (HER) 2 antibodies, pertuzumab and trastuzumab, for gastric cancer.Experimental Design: Tumor mouse xenograft models were used to examine antitumor activity. Cell proliferation was examined using crystal violet staining. HER family proteins' expression was analyzed by ELISA and immunohistochemistry. Phosphorylated proteins and heterodimers were detected by Western blotting and in situ proximity ligation assay (PLA), respectively. Apoptosis activity was examined by caspase 3/7 activity. Antibody-dependent cellular cytotoxicity (ADCC) activity was detected by xCELLigence. Microvessel density was examined by CD31 staining.Results: Pertuzumab in combination with trastuzumab showed significant antitumor activity compared with each monotherapy in NCI-N87, an HER2-positive human gastric cancer xenograft model. The efficacy was stronger than that of the maximum effective dose with each monotherapy. Similar antitumor activity was shown in 4-1ST, another HER2-positive gastric cancer model, but not in MKN-28, an HER2-negative model. Combining pertuzumab with trastuzumab enhanced cell growth inhibition and apoptosis activity by inhibiting EGFR-HER2 heterodimerization and the phosphorylation of these receptors and their downstream factors. This effect was also seen in HER2-HER3 signaling. Furthermore, pertuzumab in combination with trastuzumab potentiated the ADCC activity of those antibodies and reduced tumor microvessel density.Conclusions: We showed the significantly enhanced efficacy of pertuzumab combining with trastuzumab for HER2 overexpressing gastric cancer through the potentiation of cell growth inhibition, apoptosis activity, cell killing activity by ADCC, and antiangiogenic activity. This study suggests the clinical benefit of combination therapy with pertuzumab and trastuzumab for patients with HER2-positive gastric cancers. Clin Cancer Res; 17(15); 5060-70. Ó2011 AACR.

Pertuzumab exerts potent antitumor activity in BTC cells co-overexpressing HER2 and HER3. Pertuzumab provides a new therapeutic option against BTC.

BackgroundAccurate and reliable assessment of human epidermal growth factor receptor type 2 (HER2) status is important for selecting patients with gastric cancer who may benefit from trastuzumab treatment. Here we examined the impact of formalin fixing conditions on HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in xenografted tumor tissues.MethodsXenografted tumor tissues of the human gastric cancer cell lines NCI-N87, SCH, and SNU-16 were collected and kept at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h or 5, 7, or 10 days and embedded in paraffin. Use of 10 % NBF, 20 % NBF, or nonbuffered formalin as fixative was investigated.ResultsThe HER2 IHC scores for NCI-N87, SCH, and SNU-16 tumors were 3+, 2+, and 1+, respectively, when specimens were fixed with 10 % NBF for 24 h immediately after resection of the tumors. Specimens left for longer than 6 h before fixation had shrinkage of the tumor periphery and decreased immunostaining intensity in this region in all specimens. In SCH and SNU-16 specimens, starting fixation 24 h after tumor tissue collection induced autolysis and reduction of the number of stained cells, and 10-day-fixation lowered the HER2 score. Prolongation of fixation time did not affect FISH results, but if samples were left for more than 6 h before fixation, the FISH score was strongly reduced in SCH specimens (2.3 to 1.3). Reduced IHC staining intensity was observed with 20 % NBF and nonbuffered formalin compared to 10 % NBF.ConclusionsThe time to and length of fixation of tumor specimens can affect HER2 IHC and FISH scores. The fixative used can affect IHC results.

Human epidermal growth factor receptor 2 (HER2)-targeted therapy by trastuzumab has become increasingly important for treating HER2-positive cancers, and trastuzumab emtansine (T-DM1) is expected to serve as an effective alternative to trastuzumab. Pertuzumab, a HER2 dimerization inhibitor, showed prolonged progression-free survival when used with trastuzumab for HER2-positive breast cancer. In this study, we investigated the effect of combining T-DM1 and pertuzumab on xenografted gastric tumors. T-DM1 as a single agent showed significant antitumor activity in all the three HER2-high expression tumor models tested (NCI-N87, SCH and 4-1ST) but was ineffective against two HER2-low expression tumors (SNU-16 and MKN-28). Using the T-DM1-sensitive NCI-N87 model, the combination efficacy of T-DM1 and pertuzumab was elucidated. The combination induced significant tumor regression, whereas T-DM1 or pertuzumab alone did not. In cultured NCI-N87 cells stimulated with epidermal growth factor (EGF) or heregulin-α, concomitant treatment of T-DM1 and pertuzumab significantly inhibited proliferation and increased caspase 3/7 activity compared to either agent alone. Only the combination significantly inhibited the phosphorylation of EGFR or HER3, and its downstream factor AKT. Suppressed HER3 phosphorylation by the combination was also seen in the NCI-N87 xenografted tumors. Compared to single agent treatments, the combination treatment significantly enhanced antibody-dependent cellular cytotoxicity (ADCC) against NCI-N87 cells. These findings suggest that T-DM1 in combination with pertuzumab shows significant antitumor activity by increasing AKT signal inhibition and ADCC in HER2-positive gastric cancers.

BackgroundBevacizumab is a humanized monoclonal antibody to human vascular endothelial cell growth factor (VEGF) and has been used for many types of cancers such as colorectal cancer, non-small cell lung cancer, breast cancer, and glioblastoma. Bevacizumab might be effective against gastric cancer, because VEGF has been reported to be involved in the development of gastric cancer as well as other cancers. On the other hand, there are no established biomarkers to predict the bevacizumab efficacy in spite of clinical needs. Therefore, we tried to identify the predictive markers for efficacy of bevacizumab in gastric cancer patients by using bevacizumab-sensitive and insensitive tumor models.MethodsNine human gastric and two colorectal cancer mouse xenografts were examined for their sensitivity to bevacizumab. We examined expression levels of angiogenic factors by ELISA, bioactivity of VEGF by phosphorylation of VEGFR2 in HUVEC after addition of tumor homogenate, tumor microvessel density by CD31-immunostaining, and polymorphisms of the VEGF gene by HybriProbe™ assay.ResultsOf the 9 human gastric cancer xenograft models used, GXF97, MKN-45, MKN-28, 4-1ST, SC-08-JCK, and SC-09-JCK were bevacizumab-sensitive, whereas SCH, SC-10-JCK, and NCI-N87 were insensitive. The sensitivity of the gastric cancer model to bevacizumab was not related to histological type or HER2 status. All tumors with high levels of VEGF were bevacizumab-sensitive except for one, SC-10-JCK, which had high levels of VEGF. The reason for the refractoriness was non-bioactivity on the phosphorylation of VEGFR2 and micro-vessel formation of VEGF, but was not explained by the VEGF allele or VEGF165b. We also examined the expression levels of other angiogenic factors in the 11 gastrointestinal tumor tissues. In the refractory models including SC-10-JCK, tumor levels of another angiogenic factor, bFGF, were relatively high. The VEGF/bFGF ratio correlated more closely with sensitivity to bevacizumab than with the VEGF level.ConclusionsVEGF levels and VEGF/bFGF ratios in tumors were related to bevacizumab sensitivity of the xenografts tested. Further clinical investigation into useful predictive markers for bevacizumab sensitivity is warranted.

In a Phase III trial for HER2-positive breast cancer (the CLEOPATRA study), the triple-drug combination arm of pertuzumab plus trastuzumab plus docetaxel showed significantly longer progression-free survival and overall survival than did the trastuzumab plus docetaxel arm. In this study, we investigated the mechanism of action of the triple-drug combination therapy in vivo. For this purpose, we established a mouse xenograft model using KPL-4, a HER2-positive human breast cancer cell line, in which the triple-drug combination treatment dramatically induced tumor regression compared with double-drug combinations (trastuzumab plus docetaxel, pertuzumab plus docetaxel, or pertuzumab plus trastuzumab). Four days after the triple-drug treatment was started, strong reduction in the phosphorylation of HER2, epidermal growth factor receptor (EGFR), HER3, extracellular signal-regulated kinase (ERK), and AKT in tumor tissues was seen, despite only weak suppression of phosphorylation seen with the single- or double-drug treatments. Histopathological analysis and flow cytometric analysis showed that the triple-drug treatment enhanced apoptosis after mitotic arrest induced by docetaxel. Furthermore, infiltration of mononuclear cells around the tumor cells was strongly induced by the triple-drug combination treatment. These results suggested that the mechanism underlying the synergistic efficacy of the triple-drug combination was attributable, at least in part, to the docetaxel-mediated apoptosis being promoted by enhanced inhibition of HER2-HER3-AKT signaling as well to the intratumor infiltration of mononuclear cells induced by anti-HER2 antibodies being enhanced by docetaxel.

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