Description: Purpose: In other cancer types, HPV infection has been reported to coincide with overexpression of HER2 (ERBB2) and HER3 (ERBB3); however, the association between HER2 or HER3 expression and dimer formation in HNSCC has not been reported. Overexpression of HER2 and HER3 may contribute to resistance to EGFR inhibitors, including cetuximab, although the contribution of HPV in modulating cetuximab response remains unknown. Determination of heterodimerization of HER receptors is challenging and has not been reported in HNSCC. The present study aimed to determine the expression of HER proteins in HPV + versus HPV – HNSCC tumors using a proximity-based protein expression assay (VeraTag), and to determine the efficacy of HER-targeting agents in HPV + and HPV – HNSCC cell lines. Experimental Design: Expression of total HER1, HER2, and HER3, p95HER2, p-HER3, HER1:HER1 homodimers, HER2:HER3 heterodimers, and the HER3–PI3K complex in 88 HNSCC was determined using VeraTag, including 33 baseline tumors from individuals treated in a trial including cetuximab. Inhibition of cell growth and protein activation with cetuximab and afatinib was compared in HPV + and HPV – cetuximab-resistant cell lines. Results: Expression of total HER2, total HER3, HER2:HER3 heterodimers, and the HER3:PI3K complex were significantly elevated in HPV + HNSCC. Total EGFR was significantly increased in HPV – HNSCC where VeraTag assay results correlated with IHC. Afatinib significantly inhibited cell growth when compared with cetuximab in the HPV + and HPV – cetuximab-resistant HNSCC cell lines. Conclusions: These findings suggest that agents targeting multiple HER proteins may be effective in the setting of HPV + HNSCC and/or cetuximab resistance. Clin Cancer Res; 21(20); 4597–606. ©2015 AACR .

Description: Purpose: Non–small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. Patients with NSCLC with EGFR-activating mutation benefit greatly by gefitinib, an EGFR tyrosine kinase inhibitor. However, acquired resistance limits its clinical use. Histone deacetylases (HDAC) are oncoproteins associated with cancer progression and drug resistance. Here, we disclosed that inhibition of HDAC1 induced protein phosphatase DUSP1 upregulation to overcome gefitinib-acquired resistance. Experimental Design: The effect of HDAC1 inhibition restored gefitinib sensitivity was assessed by in vitro MTT and apoptotic assays, and in vivo xenograft and orthotopic lung cancer mouse models. Protein phosphatase array was used to detect DUSP1 expression. Immunohistochemical staining and quantitative PCR were used to analyze DUSP1 expression in clinical NSCLC specimens. Results: Gefitinib-resistant NSCLC cells showed HDAC1 overexpression, and its knockdown sensitized resistant cells to gefitinib in vitro and in preclinical models through DUSP1 expression. Overexpression of DUSP1 in resistant cells restored gefitinib sensitivity by inhibiting EGFR signaling and inducing apoptosis, whereas its knockdown in sensitive cells conferred gefitinib resistance. A novel HDAC inhibitor, WJ-26210-2, in combination with gefitinib upregulated DUSP1 expression to exert in vitro and in vivo synergistic effect on inactivation of EGFR signaling, growth inhibition, and apoptosis. Clinically, high DUSP1 level was correlated with delayed emergence of gefitinib-acquired resistance. Conclusions: Decreased DUSP1 might be a mechanism responsible for gefitinib resistance, and DUSP1 might be a biomarker for gefitinib efficacy. HDAC1 inhibition–induced DUSP1 upregulation could be a promising strategy to overcome gefitinib-acquired resistance. Clin Cancer Res; 21(2); 428–38. ©2015 AACR .

Description: Purpose: Non–small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK + ) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. Experimental Design: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined. Results: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK + cell lines, brigatinib inhibited native ALK (IC 50 , 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK + tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Conclusions: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK + , crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527–38. ©2016 AACR .

Description: Purpose: Epithelial and endothelial tyrosine kinase (Etk), also known as bone marrow X kinase (Bmx), was found to be critical in modulating the chemoresistance of small-cell lung cancer (SCLC) in our preliminary study. However, the molecular mechanisms of Etk in SCLC chemoresistance remain poorly understood. Experimental Design: We determined correlation of Etk with autophagy in SCLC. And direct inhibition of autophagy was performed to validate its effect on chemoresistance. Coimmunoprecipitation (co-IP) and GST-pull down experiments were conducted to verify the interaction of Etk and PFKFB4, after a microarray analysis. In vitro and in vivo gain or loss-of-function analyses and evaluation of PFKFB4 expression in SCLC specimens, were done to validate its role in chemoresistance. Ibrutinib was administrated in SCLC cells to verify its synergistic anti-tumor effect with chemotherapy using preclinical models including a PDX model. Results: Downregulation of Etk suppressed autophagy in chemoresistant SCLC cells, and direct inhibition of autophagy sensitized cells to chemotherapy. PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4) was identified as a downstream target of Etk and an Etk-interacting protein, which promoted chemoresistance in SCLC and was associated with poor therapeutic response and prognosis. Furthermore, ibrutinib was found to exhibit a synergistic anti-tumor effect with chemotherapy in targeting Etk. Conclusions: Our results demonstrated for the first time that Etk interacts with PFKFB4 to promote SCLC chemoresistance through regulation of autophagy. Aberrant Etk and PFKFB4 can be predictive factors for the chemotherapy response as well as potential therapeutic targets in SCLC. Clin Cancer Res; 24(4); 950–62. ©2017 AACR .

Description: Purpose: Previous studies revealed diverging results regarding the role of survivin in squamous cell carcinoma of the head and neck (SCCHN). This study aimed to evaluate the clinical significance of survivin expression in SCCHN; the function of survivin in DNA-damage repair following ionizing radiation therapy (RT) in SCCHN cells; and the potential of honokiol to enhance RT through downregulation of survivin. Experimental Design: Expression of survivin in SCCHN patient primary tumor tissues ( n = 100) was analyzed and correlated with clinical parameters. SCCHN cell lines were used to evaluate the function of survivin and the effects of honokiol on survivin expression in vitro and in vivo . Results: Overexpression of survivin was significantly associated with lymph nodes' metastatic status ( P = 0.025), worse overall survival (OS), and disease-free survival (DFS) in patients receiving RT ( n = 65, OS: P = 0.024, DFS: P = 0.006) and in all patients with SCCHN ( n = 100, OS: P = 0.002, DFS: P = 0.003). In SCCHN cells, depletion of survivin led to increased DNA damage and cell death following RT, whereas overexpression of survivin increased clonogenic survival. RT induced nuclear accumulation of survivin and its molecular interaction with -H2AX and DNA-PKCs. Survivin specifically bound to DNA DSB sites induced by I-SceI endonuclease. Honokiol (which downregulates survivin expression) in combination with RT significantly augmented cytotoxicity in SCCHN cells with acquired radioresistance and inhibited growth in SCCHN xenograft tumors. Conclusions: Survivin is a negative prognostic factor and is involved in DNA-damage repair induced by RT. Targeting survivin using honokiol in combination with RT may provide novel therapeutic opportunities. Clin Cancer Res; 24(4); 858–69. ©2017 AACR .

Description: Purpose: Expression of p95HER2 (p95), a truncated form of the HER2 receptor, which lacks the trastuzumab binding site but retains kinase activity, has been reported as a prognostic biomarker for poor outcomes in patients with trastuzumab-treated HER2-positive metastatic breast cancer. The impact of p95 expression on trastuzumab treatment efficacy in early HER2-positive breast cancer is less clear. In the current study, p95 was tested as a predictive marker of trastuzumab treatment benefit in the HER2-positive subset of the FinHer adjuvant phase III trial. Experimental Design: In the FinHer trial, 232 patients with HER2-positive early breast cancer were randomized to receive chemotherapy plus 9 weeks of trastuzumab or no trastuzumab treatment. Quantitative p95 protein expression was measured in formalin-fixed paraffin-embedded samples using the p95 VeraTag assay (Monogram Biosciences), specific for the M611 form of p95. Quantitative HER2 protein expression was measured using the HERmark assay (Monogram Biosciences). Distant disease-free survival (DDFS) was used as the primary outcome measure. Results: In the arm receiving chemotherapy only, increasing log 10 (p95) correlated with shorter DDFS (HR, 2.0; P = 0.02). In the arm receiving chemotherapy plus trastuzumab ( N = 95), increasing log 10 (p95) was not correlated with a shorter DDFS. In a combined analysis of both treatment arms, high breast tumor p95 content was significantly correlated with trastuzumab treatment benefit in multivariate models (interaction P = 0.01). Conclusions: A high p95HER2/HER2 ratio identified patients with metastatic breast cancer with poor outcomes on trastuzumab-based therapies. Further investigation of the p95HER2/HER2 ratio as a potential prognostic or predictive biomarker for HER2-targeted therapy is warranted. Clin Cancer Res; 24(13); 3046–52. ©2018 AACR .

Description: Purpose: p95HER2 is a truncated form of HER2 that confers resistance to trastuzumab in vitro , but clinical results have been conflicting to date. Given that p95HER2 levels correlate with total HER2 expression levels, which confer better outcomes, we sought to evaluate the p95HER2/HER2 ratio in the North Central Cancer Treatment Group N0337 and N98-32-52 trials. Experimental Design: The HERmark assay and VeraTag technology (Monogram Biosciences) were used to measure total HER2 and p95HER2 expression levels in 91 patient samples. Results: In the multivariate model, increasing total HER2 level was significantly associated with longer (OS; HR, 0.33; P = 0.002) and decreasing p95HER2 level was significantly associated with longer OS (HR, 4.2; P = 0.01). Total HER2 expression level was significantly associated with longer progression-free survival (PFS) (HR, 0.57; P = 0.04), whereas p95HER2 level was not (HR, 1.7; P = 0.25). However, there was a positive association between p95HER2 and total HER2 expression levels ( R 2 = 0.48; P 〈 0.001). Consistent with our hypothesis, the ratio of p95HER2/HER2 was significantly associated with worsening PFS (HR, 1.7; P = 0.04) and OS (HR, 2.8; P = 0.002). Patients with the highest tertile of p95HER2/HER2 values had significantly less favorable PFS (HR, 1.8; P = 0.06) and OS (HR, 2.3; P = 0.02). Conclusions: A high p95HER2/HER2 ratio identified patients with metastatic breast cancer with poor outcomes on trastuzumab-based therapies. Further investigation of the p95HER2/HER2 ratio as a potential prognostic or predictive biomarker for HER2-targeted therapy is warranted. Clin Cancer Res; 24(13); 3053–8. ©2018 AACR .

Description: Purpose: Current tumor–node–metastasis (TNM) staging system cannot provide adequate information for prediction of prognosis and chemotherapeutic benefits. We constructed a classifier to predict prognosis and identify a subset of patients who can benefit from adjuvant chemotherapy. Experimental Design: We detected expression of 15 immunohistochemistry (IHC) features in tumors from 251 gastric cancer (GC) patients and evaluated the association of their expression level with overall survival (OS) and disease-free survival (DFS). Then, integrating multiple clinicopathologic features and IHC features, we used support vector machine (SVM)–based methods to develop a prognostic classifier (GC-SVM classifier) with features. Further validation of the GC-SVM classifier was performed in two validation cohorts of 535 patients. Results: The GC-SVM classifier integrated patient sex, carcinoembryonic antigen, lymph node metastasis, and the protein expression level of eight features, including CD3 invasive margin (IM) , CD3 center of tumor (CT) , CD8 IM , CD45RO CT , CD57 IM , CD66b IM , CD68 CT , and CD34. Significant differences were found between the high- and low-GC-SVM patients in 5-year OS and DFS in training and validation cohorts. Multivariate analysis revealed that the GC-SVM classifier was an independent prognostic factor. The classifier had higher predictive accuracy for OS and DFS than TNM stage and can complement the prognostic value of the TNM staging system. Further analysis revealed that stage II and III GC patients with high-GC-SVM were likely to benefit from adjuvant chemotherapy. Conclusions: The newly developed GC-SVM classifier was a powerful predictor of OS and DFS. Moreover, the GC-SVM classifier could predict which patients with stage II and III GC benefit from adjuvant chemotherapy. Clin Cancer Res; 24(22); 5574–84. ©2018 AACR .

Description: Some patients with soft-tissue sarcoma (STS) report a history of injury at the site of their tumor. Although this phenomenon is widely reported, there are relatively few experimental systems that have directly assessed the role of injury in sarcoma formation. We recently described a mouse model of STS whereby p53 is deleted and oncogenic Kras is activated in muscle satellite cells via a Pax7CreER driver following intraperitoneal injection with tamoxifen. Here, we report that after systemic injection of tamoxifen, the vast majority of Pax7-expressing cells remain quiescent despite mutation of p53 and Kras. The fate of these muscle progenitors is dramatically altered by tissue injury, which leads to faster kinetics of sarcoma formation. In adult muscle, quiescent satellite cells will transition into an active state in response to hepatocyte growth factor (HGF). We show that modulating satellite cell quiescence via intramuscular injection of HGF increases the penetrance of sarcoma formation at the site of injection, which is dependent on its cognate receptor c-MET. Unexpectedly, the tumor-promoting effect of tissue injury also requires c-Met. These results reveal a mechanism by which HGF/c-MET signaling promotes tumor formation after tissue injury in a mouse model of primary STS, and they may explain why some patients develop a STS at the site of injury. Cancer Res; 75(3); 605–14. ©2014 AACR.

Description: As anticancer therapies designed to target specific molecular pathways have been developed, it has become critical to develop methods to assess the response induced by such agents. Although traditional, anatomic CT, and MRI examinations are useful in many settings, increasing evidence suggests that these methods cannot answer the fundamental biologic and physiologic questions essential for assessment and, eventually, prediction of treatment response in the clinical trial setting, especially in the critical period soon after treatment is initiated. To optimally apply advances in quantitative imaging methods to trials of targeted cancer therapy, new infrastructure improvements are needed that incorporate these emerging techniques into the settings where they are most likely to have impact. In this review, we first elucidate the needs for therapeutic response assessment in the era of molecularly targeted therapy and describe how quantitative imaging can most effectively provide scientifically and clinically relevant data. We then describe the tools and methods required to apply quantitative imaging and provide concrete examples of work making these advances practically available for routine application in clinical trials. We conclude by proposing strategies to surmount barriers to wider incorporation of these quantitative imaging methods into clinical trials and, eventually, clinical practice. Our goal is to encourage and guide the oncology community to deploy standardized quantitative imaging techniques in clinical trials to further personalize care for cancer patients and to provide a more efficient path for the development of improved targeted therapies. Clin Cancer Res; 22(2); 284–90. ©2016 AACR .