Naoki Takegawa

HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer

Background Patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS benefit from epidermal growth factor receptor (EGFR)-targeted therapy. However, patients who are treated with anti-EGFR antibodies will eventually develop the resistance to those agents. HER2 amplification is one of the mechanisms conferring resistance to anti-EGFR antibody therapy and could therefore be a potential therapeutic target. The aim of this study was to detect HER2 amplification in circulating tumor DNA (ctDNA) from patients with CRC and acquired resistance to anti-EGFR antibody therapy. Results Our data showed that 22% (4/18) of patients in the cohort exhibited HER2 amplification. One of these patients was found to be positive for HER2 amplification in matched tumor specimens collected after cetuximab therapy, at which point the patient had acquired cetuximab resistance, despite being negative for HER2 amplification prior to therapy. Methods We analyzed plasma ctDNA using digital polymerase chain reaction (PCR) from 18 patients with CRC, who had been treated with anti-EGFR antibody-based therapy (cetuximab) and subsequently acquired resistant cetuximab. HER2 gene copy number was analyzed using fluorescence in situ hybridization in tumor samples before and after acquisition of resistance to cetuximab-based therapy. Conclusion Analysis of plasma ctDNA by digital PCR could be useful for detecting HER2 amplification in patients with CRC who were resistant to anti-EGFR antibody therapy.

DS-8201a, a new HER2-targeting antibody–drug conjugate incorporating a novel DNA topoisomerase I inhibitor, overcomes HER2-positive gastric cancer T-DM1 resistance

Anti‐HER2 therapies are beneficial for patients with HER2‐positive breast or gastric cancer. T‐DM1 is a HER2‐targeting antibody–drug conjugate (ADC) comprising the antibody trastuzumab, a linker, and the tubulin inhibitor DM1. Although effective in treating advanced breast cancer, all patients eventually develop T‐DM1 resistance. DS‐8201a is a new ADC incorporating an anti‐HER2 antibody, a newly developed, enzymatically cleavable peptide linker, and a novel, potent, exatecan‐derivative topoisomerase I inhibitor (DXd). DS‐8201a has a drug‐to‐antibody‐ratio (DAR) of 8, which is higher than that of T‐DM1 (3.5). Owing to these unique characteristics and unlike T‐DM1, DS‐8201a is effective against cancers with low‐HER2 expression. In the present work, T‐DM1‐resistant cells (N87‐TDMR), established using the HER2‐positive gastric cancer line NCI‐N87 and continuous T‐DM1 exposure, were shown to be susceptible to DS‐8201a. The ATP‐binding cassette (ABC) transporters ABCC2 and ABCG2 were upregulated in N87‐TDMR cells, but HER2 overexpression was retained. Furthermore, inhibition of ABCC2 and ABCG2 by MK571 restored T‐DM1 sensitivity. Therefore, resistance to T‐DM1 is caused by efflux of its payload DM1, due to aberrant expression of ABC transporters. In contrast to DM1, DXd payload of DS‐8201a inhibited the growth of N87‐TDMR cells in vitro. This suggests that either DXd may be a poor substrate of ABCC2 and ABCG2 in comparison to DM1, or the high DAR of DS‐8201a relative to T‐DM1 compensates for increased efflux. Notably, N87‐TDMR xenograft tumor growth was prevented by DS‐8201a. In conclusion, the efficacy of DS‐8201a as a treatment for patients with T‐DM1‐resistant breast or gastric cancer merits investigation.

T790M-selective EGFR-TKI combined with dasatinib as an optimal strategy for overcoming EGFR-TKI resistance in T790M-positive non-small cell lung cancer

T790M mutation–selective EGFR tyrosine kinase inhibitors (EGFR-TKI) have demonstrated clinical benefits in non–small cell lung cancer (NSCLC) patients harboring T790M mutation, which is the major cause of resistance to EGFR-TKI. However, their efficacy is limited, possibly due to the emergence of apoptosis resistance in T790M-positive NSCLC. We previously identified Src family kinases as cooncogenic drivers along with T790M and found that the Src inhibitor dasatinib combined with an irreversible or a preclinical T790M-selective EGFR-TKI enhanced antitumor activity in T790M-positive cells. In the current study, we evaluated the efficacy of dasatinib combined with the clinically relevant T790M-selective EGFR-TKI ASP8273 or osimertinib in EGFR mutation–positive NSCLC with or without T790M mutation. A cell viability assay revealed that dasatinib had synergistic effects with these TKIs in T790M-positive cells and simultaneously inhibited Src, Akt, and Erk, which remained activated upon single-agent treatment. Dasatinib also increased the rate of apoptosis in T790M-positive cells induced by T790M-selective EGFR-TKIs, as determined by the Annexin-V binding assay; this was associated with downregulation of the antiapoptotic Bcl-2 family member Bcl-xL, a finding that was confirmed in mice bearing T790M-positive xenografts. Our results suggest that Bcl-xL plays a key role in the apoptosis resistance of T790M-positive NSCLC, and that dasatinib combined with clinically relevant T790M-selective EGFR-TKIs is potentially effective in overcoming resistance to first-generation EGFR-TKIs in NSCLC patients with acquired T790M. Mol Cancer Ther; 16(11); 2563–71. ©2017 AACR.

Heregulin-expressing HER2-positive breast and gastric cancer exhibited heterogeneous susceptibility to the anti-HER2 agents lapatinib, trastuzumab and T-DM1

Background Overexpression of heregulin, a HER3 ligand, is one mechanism that confers resistance to the anti-HER2 agents trastuzumab and lapatinib. We investigated the impact of heregulin expression on the efficacy of HER2-targeted therapeutic agents, including trastuzumab, trastuzumab emtansine (T-DM1) and lapatinib, in vitro and in vivo and evaluated the heregulin messenger RNA (mRNA) levels in specimens from patients with HER2-positive breast or gastric cancer. Results Cell proliferation and apoptosis assays demonstrated that heregulin conferred robust resistance to lapatinib and trastuzumab via HER3-Akt pathway activation followed by survivin overexpression; however, heregulin conferred minimal or no resistance to T-DM1 and paclitaxel. The heregulin mRNA level of one of 10 patients was up-regulated after the acquisition of resistance to trastuzumab-based therapy. Materials and Methods SK-BR-3, NCI-N87, BT-474, MDA-MB-453, HCC1954, SNU-216 and 4-1ST cells were pharmacologically treated with recombinant heregulin or transfected with the heregulin gene. We also assessed the expression of heregulin mRNA in HER2-positive breast or gastric cancer samples before and after trastuzumab-based therapy using a RT-PCR-based method. Conclusions mRNA up-regulation of heregulin was observed in clinical breast cancer specimens during trastuzumab-based treatment, but heregulin overexpression had a limited effect on the sensitivity to T-DM1 in vitro and in vivo.

Combined Analysis of Plasma Amphiregulin and Heregulin Predicts Response to Cetuximab in Metastatic Colorectal Cancer

Background Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), is associated with the efficacy of cetuximab, an antibody against EGFR, as treatment for colorectal cancer (CRC). In contrast, the HER3 ligand heregulin correlates with cetuximab resistance. In this study, we evaluated how the combined levels of circulating amphiregulin and heregulin affect clinical outcomes in patients who receive cetuximab as therapy against advanced CRC. Methods Plasma levels of amphiregulin and heregulin were measured by enzyme-linked immunosorbent assay in 50 patients with CRC in a training cohort, and in 10 patients in a validation cohort. The combined expression was then assessed with clinical outcome after receiver operating characteristics analysis. Results Overall response rate was 26%, and median progression-free survival was 110 days in the training cohort. Patients with high amphiregulin and low heregulin had significantly higher objective response rate at 58% and significantly longer progression-free survival of 216 days. This result was confirmed in the validation cohort. Conclusion A subgroup of CRC patients with high amphiregulin and low heregulin respond to cetuximab therapy better than other patients.

HER2 as an Emerging Oncotarget for Colorectal Cancer Treatment After Failure of Anti-Epidermal Growth Factor Receptor Therapy

Abstract Amplification of the HER2 gene is an indicator of poor prognosis for several kinds of malignancies such as breast and gastric cancer, and anti‐HER2 targeting therapies provide clinical benefits in these patients. In 2011, HER2 was identified as a resistance molecule for de novo and secondary anti‐epidermal growth factor receptor (EGFR) antibody therapy. HER2 activation provides a bypass signaling pathway after anti‐EGFR antibody treatment of colorectal cancer. Cell line‐based screening revealed that HER2 genomic amplification induces resistance to the anti‐EGFR antibody cetuximab in colorectal cancer. Recently, HER2 itself has been recognized as a target for oncotherapy in colorectal cancer. The first part of this review provides an update on the present state of knowledge about the role of HER2 in colorectal cancer, including its prognostic relevance and role in resistance to anti‐EGFR antibody treatment. In the second part of the review, we discuss the results of preclinical and clinical studies that examined the potential utility of anti‐HER2 targeted therapy in colorectal cancer. Although it acts as a barrier for other molecular targeting agents such as cetuximab, HER2 itself is a promising target for oncotherapy. Current research indicates that anti‐HER2 drugs will be developed further and introduced into clinical practice for the treatment of patients with HER2‐positive colorectal cancer.

B7-H3 Negatively Modulates CTL-Mediated Cancer Immunity

Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting. Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry. Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction. Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

Clinical evaluation of palliative chemoradiotherapy for metastatic esophageal cancer

Platinum-based chemotherapy is considered a standard treatment option for patients with metastatic esophageal carcinoma. However, the overall survival of patients receiving such treatment is <1 year. A common presenting symptom of esophageal cancer is dysphagia, which has a substantial impact on quality of life. We have now retrospectively evaluated the efficacy and safety of palliative chemoradiotherapy for patients with stage IV esophageal cancer, most of whom are unfit for curative chemoradiotherapy. Fifty consecutive patients diagnosed with stage IV esophageal cancer were treated with concurrent chemoradiotherapy at Kindai University Hospital between April 2008 and December 2014. Most (90%) patients received a total radiation dose of at least 50 Gy, and the median number of treatment cycles per patient was four for the combination of 5-fluorouracil and cisplatin. The response of the primary tumor and the overall response were 80% and 44%, respectively. The dysphagia score was improved after chemoradiotherapy in 36 (72%) patients and did not change between before and after treatment in 14 (28%) patients. With a median follow-up time of 9.4 months from the start of chemoradiotherapy, the median progression-free survival and overall survival were 4.7 and 12.3 months, respectively. Three patients (T4b in two, T3 in one) developed esophagobronchial fistula after completion of chemoradiotherapy (n = 2) or after disease progression (n = 1), resulting in death in each case. Our results suggest that palliative chemoradioiotherapy was safe and contributed the improvement of dysphagia in patients with stage IV esophageal cancer.Platinum-based chemotherapy is considered a standard treatment option for patients with metastatic esophageal carcinoma. However, the overall survival of patients receiving such treatment is <1 year. A common presenting symptom of esophageal cancer is dysphagia, which has a substantial impact on quality of life. We have now retrospectively evaluated the efficacy and safety of palliative chemoradiotherapy for patients with stage IV esophageal cancer, most of whom are unfit for curative chemoradiotherapy. Fifty consecutive patients diagnosed with stage IV esophageal cancer were treated with concurrent chemoradiotherapy at Kindai University Hospital between April 2008 and December 2014. Most (90%) patients received a total radiation dose of at least 50 Gy, and the median number of treatment cycles per patient was four for the combination of 5-fluorouracil and cisplatin. The response of the primary tumor and the overall response were 80% and 44%, respectively. The dysphagia score was improved after chemoradiotherapy in 36 (72%) patients and did not change between before and after treatment in 14 (28%) patients. With a median follow-up time of 9.4 months from the start of chemoradiotherapy, the median progression-free survival and overall survival were 4.7 and 12.3 months, respectively. Three patients (T4b in two, T3 in one) developed esophagobronchial fistula after completion of chemoradiotherapy (n = 2) or after disease progression (n = 1), resulting in death in each case. Our results suggest that palliative chemoradioiotherapy was safe and contributed the improvement of dysphagia in patients with stage IV esophageal cancer.

Abstract 509: HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer

Background: Patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS benefit from epidermal growth factor receptor (EGFR)-targeted therapy. However, patients who are treated with anti-EGFR antibodies will eventually develop the resistance to those agents. We previously found that HER2 amplification was one of the mechanisms conferring resistance to anti-EGFR antibody therapy and could therefore be a potential therapeutic target (Yonesaka et al, STM 2011). The aim of this study was to detect HER2 amplification in circulating tumor DNA (ctDNA) from patients with CRC and acquired resistance to anti-EGFR antibody therapy. Methods: We analyzed plasma ctDNA using digital polymerase chain reaction (PCR) from 18 patients with CRC, who had been treated with anti-EGFR antibody-based therapy (cetuximab) and subsequently acquired resistant cetuximab. We aimed to obtain at least 100 droplets for HER2 and EFTUD2 to accurately assess the ratio. Digital PCR data were analyzed using the QuantaSoft analytical software package (Bio-Rad). The copy number concentration of each gene (HER2 and EFTUD2) was estimated from the Poisson distribution. HER2 amplification with digital PCR was defined as a HER2 ratio (HER2/EFTUD2 copy number ratio) of 1.25 accordingly (Gevensleven H et al, CCR 2013). HER2 gene copy number was analyzed using fluorescence in situ hybridization also in tumor samples before and after acquisition of resistance to cetuximab-based therapy. Results: Our data showed various HER2 copy number in plasma ctDNA from CRC patients (median: 1.09; range: 0.94-5.18). 22% (4/18) of patients in the cohort exhibited HER2 amplification. One of these patients was found to be positive for HER2 amplification in matched tumor specimens collected after cetuximab therapy, at which point the patient had acquired cetuximab resistance, despite being negative for HER2 amplification prior to therapy (pre: 1.14, post: 2.78). Conclusion: Analysis of plasma ctDNA by digital PCR could be useful for detecting HER2 amplification in patients with CRC who were resistant to anti-EGFR antibody therapy. We are now conducting another, large-scale study for evaluating HER2 amplification by ctDNA in CRC patients using digital PCR. Citation Format: NAOKI TAKEGAWA. HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 509.

Phase II Trial of 5‐Fluorouracil, Docetaxel, and Nedaplatin (UDON) Combination Therapy for Recurrent or Metastatic Esophageal Cancer

Lessons Learned. The 5‐fluorouracil, docetaxel, and nedaplatin (UDON) regimen was well tolerated and showed promising antitumor activity in terms of both objective response rate and survival for patients with advanced or recurrent esophageal squamous cell carcinoma in the first‐line setting. UDON may be an optimal treatment option for patients with advanced esophageal cancer who are unfit for docetaxel, cisplatin, and 5‐fluorouracil regimens. The high response rate as well as the rapid and marked tumor shrinkage associated with UDON suggest that further evaluation of this regimen in the neoadjuvant setting is warranted. Background. A phase II study was performed to evaluate the efficacy and safety of 5‐fluorouracil (5‐FU), docetaxel, and nedaplatin (UDON) combination therapy for untreated recurrent or metastatic esophageal cancer. Methods. Patients received intravenous nedaplatin (90 mg/m2) on day 1, docetaxel (35 mg/m2) on days 1 and 15, and 5‐fluorouracil (800 mg/m2) on days 1–5 of a 4‐week cycle. The primary endpoint was response rate, with secondary endpoints including overall survival (OS), progression‐free survival (PFS), dysphagia score, and adverse events. Results. Between March 2015 and July 2017, 23 patients were enrolled. Of 22 evaluable patients, 16 and 4 individuals experienced a partial response and stable disease, respectively, yielding a response rate of 72.7% (95% confidence interval [CI], 49.8%–89.3%) and disease control rate of 90.9% (95% CI, 70.8%–98.9%). Median OS and PFS were 11.2 months (95% CI, 9.1 months to not reached) and 6.0 months (95% CI, 2.5–10.6 months), respectively. Eleven (64.7%) of the 17 patients with a primary lesion showed amelioration of dysphagia after treatment. Frequent adverse events of grade 3 or 4 included neutropenia (87.0%) and leukopenia (39.1%). Febrile neutropenia was observed in two patients (8.7%). Conclusion. This phase II study demonstrated promising antitumor activity and good tolerability of UDON.