Nhu-An Pham

The association between EGFR variant III, HPV, p16, c-MET, EGFR gene copy number and response to EGFR inhibitors in patients with recurrent or metastatic squamous cell carcinoma of the head and neck

BackgroundWe examine the potential prognostic and predictive roles of EGFR variant III mutation, EGFR gene copy number (GCN), human papillomavirus (HPV) infection, c-MET and p16INK4Aprotein expression in recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN).MethodsWe analyzed the archival tumor specimens of 53 patients who were treated in 4 phase II trials for R/M SCCHN. Two trials involved the EGFR inhibitor erlotinib, and 2 trials involved non-EGFR targeted agents. EGFRvIII mutation was determined by quantitative RT-PCR, HPV DNA by Linear Array Genotyping, p16 and c-MET protein expression by immunohistochemistry, and EGFR GCN by FISH.ResultsEGFRvIII mutation, detected in 22 patients (42%), was associated with better disease control, but no difference was seen between erlotinib-treated versus non-erlotinib treated patients. EGFRvIII was not associated with TTP or OS. The presence of HPV DNA (38%), p16 immunostaining (32%), c-MET high expression (58%) and EGFR amplification (27%), were not associated with response, TTP or OS.ConclusionEGFRvIII mutation, present in about 40% of SCCHN, appears to be an unexpected prognostic biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Larger prospective studies are required to validate its significance.

A multicenter phase II clinical trial of lapatinib (GW572016) in hormonally untreated advanced prostate cancer.

Objectives:Lapatinib (GW572016) is a selective and potent dual tyrosine kinase inhibitor of the epidermal growth factor 1 (EGFR) and 2 (HER2), approved in the treatment of HER2 positive breast cancer. Since EGFR and HER2 overexpression has also been seen in prostate cancer and appears to correlate with a worse clinical outcome, Lapatinib may represent a novel therapeutic strategy in prostate cancer. This Phase II multicenter clinical trial is the first to evaluate Lapatinib in early stage, hormonally untreated recurrent or metastatic prostate cancer. Methods:Eligible patients received lapatinib 1500 mg PO daily until progression. The primary end point was prostate specific antigen (PSA) response. Archival tumor tissue was collected for EGFR and HER2 analysis. Results:A total of 23 patients, median age 67, ECOG PS 0–2, mean baseline PSA 7.5, were evaluable for response. In total, 125 cycles were administered. The most frequent adverse events were lymphopenia, fatigue, rash, dyspepsia, and diarrhea. Grade 3+ increased alanine aminotransferase (ALT) was reported in 2 patients, and grade 4 blurry vision in 1 patient. No PSA responses were seen. Median time to progression (TTP) was 4.6 months and 6 months progression-free estimate was 44.5%. Conclusions:Lapatinib was well tolerated but like other EGFR- and HER2-targeted agents in advanced HRPC failed to show significant antitumor activity even in this very early stage hormonally untreated population.

Clinical Utility of Patient-Derived Xenografts to Determine Biomarkers of Prognosis and Map Resistance Pathways in EGFR-Mutant Lung Adenocarcinoma

PURPOSE Although epidermal growth factor receptor (EGFR) -mutated adenocarcinomas initially have high response rates to EGFR tyrosine kinase inhibitors (TKIs), most patients eventually develop resistance. Patient-derived xenografts (PDXs) are considered preferred preclinical models to study the biology of patient tumors. EGFR-mutant PDX models may be valuable tools to study the biology of these tumors and to elucidate mechanisms of resistance to EGFR-targeted therapies. METHODS Surgically resected early-stage non-small-cell lung carcinoma (NSCLC) tumors were implanted into nonobese diabetic severe combined immune deficient (NOD-SCID) mice. EGFR TKI treatment was initiated at tumor volumes of 150 μL. Gene expression analysis was performed using a microarray platform. RESULTS Of 33 lung adenocarcinomas with EGFR activating mutations, only 6 (18%) engrafted and could be propagated beyond passage one. Engraftment was associated with upregulation of genes involved in mitotic checkpoint and cell proliferation. A differentially expressed gene set between engrafting and nonengrafting patients could identify patients harboring EGFR-mutant tumor with significantly different prognoses in The Cancer Genome Atlas Lung Adenocarcinoma datasets. The PDXs included models with variable sensitivity to first- and second-generation EGFR TKIs and the monoclonal antibody cetuximab. All EGFR-mutant NSCLC PDXs studied closely recapitulated their corresponding patient tumor phenotype and clinical course, including response pattern to EGFR TKIs. CONCLUSION PDX models closely recapitulate primary tumor biology and clinical outcome. They may serve as important laboratory models to investigate mechanisms of resistance to targeted therapies, and for preclinical testing of novel treatment strategies.

Integrin α11β1 regulates cancer stromal stiffness and promotes tumorigenicity and metastasis in non-small cell lung cancer

Integrin α11β1 is a stromal cell-specific receptor for fibrillar collagens and is overexpressed in carcinoma-associated fibroblasts (CAFs). We have investigated its direct role in cancer progression by generating severe combined immune deficient (SCID) mice deficient in integrin α11 (α11) expression. The growth of A549 lung adenocarcinoma cells and two patient-derived non-small cell lung carcinoma (NSCLC) xenografts in these α11 knockout (α11−/−) mice was significantly impeded, as compared with wild-type (α11+/+) SCID mice. Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11−/− and α11+/+ mice showed significant reduction in the metastatic potential of these cells in the α11−/− mice. We identified that collagen cross-linking is associated with stromal α11 expression, and the loss of tumor stromal α11 expression was correlated with decreased collagen reorganization and stiffness. This study shows the role of integrin α11β1, a receptor for fibrillar collagen in differentiation of fibroblasts into CAFs. Furthermore, our data support an important role for α11 signaling pathway in CAFs, promoting tumor growth and metastatic potential of NSCLC cells and being closely associated with collagen cross-linking and the organization and stiffness of fibrillar collagen matrices.

Differential roles of cyclin D1 and D3 in pancreatic ductal adenocarcinoma.

BackgroundThe cyclin D1 (CCND1) and cyclin D3 (CCND3) are frequently co-overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here we examine their differential roles in PDAC.ResultsCCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p < 0.005). In contrast, focal adhesion/actin cytoskeleton, MAPK and NF B signaling appeared to characterize the target genes and their interacting proteins in CCND1 suppressed PANC1 cells.ConclusionsOur results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis.

Dissociation of gemcitabine sensitivity and protein kinase B signaling in pancreatic ductal adenocarcinoma models.

Objective: To understand the impact of protein kinase B (PKB; Akt) signaling on growth and protection from apoptosis in pancreatic ductal adenocarcinoma models demonstrating differences in PKB activity. Methods: Gemcitabine sensitivity was investigated in a panel of cell lines, characterized by differences in levels of activated PKB. Suppression of PKB activity was achieved with an inhibitor of phosphatidylinositol 3-kinase (wortmannin) and silencing RNA. Results: Enhanced gemcitabine (2′,2′-difluoro-2′-deoxycytidine)-induced cytotoxicity in vitro was achieved with suppression of high PKB activity with wortmannin in BxPC-3, PK-1, and PK-8 cells and silencing RNA targeted to total PKB, rather than PKB&bgr;, in PANC-1 cells. Opposite to gemcitabine sensitivity levels in vitro, the growth of PANC-1 xenografts was inhibited with gemcitabine treatment, whereas BxPC-3 became drug resistant. Monolayer cell cultures reestablished from solid tumors behaved similarly to original cultures, suggesting that the tumor microenvironment has a critical role in determining drug sensitivity. A comparison of transcript profiles of the models indicated that PKB signaling might be modulated by a number of pathways responsive to the tumor hypoxia microenvironment. Conclusions: These results suggested that gemcitabine efficacy involving the PKB pathway depends on PKB activity, its mechanisms of enhanced activity, as well as its function in a signaling network.

Association of Ipilimumab With Safety and Antitumor Activity in Women With Metastatic or Recurrent Human Papillomavirus–Related Cervical Carcinoma

Importance Based on evidence of human papillomavirus (HPV)–induced immune evasion, immunotherapy may be an attractive strategy in cervical cancer. Ipilimumab is a fully humanized monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4), which acts to downregulate the T-cell immune response. Objective To assess the safety and antitumor activity of ipilimumab in recurrent cervical cancer. Design, Setting, and Participants A multicenter trial was designed for patients with metastatic cervical cancer (squamous cell carcinoma or adenocarcinoma) with measurable disease and progression after at least 1 line of platinum chemotherapy. A run-in safety cohort using ipilimumab, 3 mg/kg, every 21 days for 4 cycles in 6 patients was followed by a phase II cohort of ipilimumab, 10 mg/kg, every 21 days for 4 cycles and then 4 cycles of maintenance therapy every 12 weeks for patients demonstrating radiologic response or stabilization. Immune correlative studies were performed on peripheral blood before and after therapy on archival tissue and fresh tumor obtained prior to registration and 7 days after cycle 2. The study was conducted from December 3, 2012, to September 15, 2014. The data were analyzed from April 2016 to June 2016 and in July 2017. Main Outcomes and Measures The primary end points were safety and objective response rate. Immune analyses were performed on blood and tumor tissue. Results A total of 42 women (median age, 49 years; range, 23-78 years) were enrolled (29 [69%] squamous cell cervical cancer and 13 [31%] adenocarcinoma; 37 [93%] of 40 patients with tissue available for analysis had HPV-positive confirmation; there was no archival tissue for 2 women). Grade 3 toxic effects included diarrhea in 4 patients, 3 of whom had colitis. Of 34 patients evaluated for best response (Response Evaluation Criteria in Solid Tumors, version 1.1), 1 patient had partial response and 10 had stable disease. The median progression-free survival and overall survival were 2.5 months (95% CI, 2.1-3.2 months) and 8.5 months (95% CI, 3.6-not reached; 1 patient was still alive), respectively. Intratumoral pretreatment CD3, CD4, CD8, FoxP3, indoleamine 2,3-dioxygenase, and programmed cell death ligand 1 (PD-L1) expression was not predictive of benefit and did not significantly change with treatment. Multicolor flow cytometry on peripheral lymphocytes revealed a treatment-dependent increase of inducible T-cell costimulator, human leukocyte antigen–antigen D related, and PD-1 during initial treatment, which returned to baseline during maintenance. Conclusions and Relevance Ipilimumab was tolerable in this population but did not show significant single-agent activity. Immune changes were induced by anti–CTLA-4 therapy but did not correlate with clinical activity. Changes in these markers may guide further treatment strategies.

Novel combinations of PI3K-mTOR inhibitors with dacomitinib or chemotherapy in PTEN-deficient patient-derived tumor xenografts

PTEN inactivation occurs commonly in human cancers and putatively activates the PI3K/AKT/ mTOR pathway. Activation of this pathway has been involved in resistance to chemotherapy or anti-EGFR/HER2 therapies. We evaluated the combination of PI3K-mTOR inhibitors with chemotherapy or the pan-HER inhibitor dacomitinib in PTEN-deficient patient-derived tumor xenografts (PDX). Three PDXs were selected for their lack of PTEN expression by immunohistochemistry: a triple-negative breast cancer (TNBC), a KRAS G12R low-grade serous ovarian cancer (LGSOC), and KRAS G12C and TP53 R181P lung adenocarcinoma (LADC). Two dual PI3K-mTOR inhibitors were evaluated—PF-04691502 and PF-05212384—in combination with cisplatin, paclitaxel, or dacomitinib. The addition of PI3K-mTOR inhibitors to cisplatin or paclitaxel increased the activity of chemotherapy in the TNBC and LGSOC models; whereas no added activity was observed in the LADC model. Pharmacodynamic modulation of pS6 and pAKT was observed in the group treated with PI3K-mTOR inhibitor. Our research suggests that the addition of a PI3K-mTOR inhibitor may enhance tumor growth inhibition when compared to chemotherapy alone in certain PTEN-deficient PDXs. However, this benefit was absent in the KRAS and TP53 mutant LADC model. The role of PTEN deficiency in the antitumor activity of these combinations should be further investigated in the clinic.

Abstract B32: Fidelity of genomic and proteomic features of patient-derived xenografts of lung cancers

The establishment of valid lung carcinoma preclinical models for testing new cancer therapies is necessary, as existing cell lines and mouse models may not recapitulate the full spectrum of heterogeneity of patient tumors. Studies suggest lung cancer patient-derived xenograft (PDX) models recapitulate well gene copy number variation, gene expression profiles, and metabolic states of corresponding patient tumors. However, an understanding of mechanisms linking cancer-associated genome, transcriptome, and proteome alterations with driver mutations and dysregulated signal transduction networks in primary and PDX models is lacking. We report a large (>150) resource of lung cancer PDX models, derived from surgically resected tumors, and endobronchial ultrasound-guided (EBUS) and CT-guided biopsies. Tumor specimens were grown and serially passaged in the subcutaneous pocket at the flanks of NSG mice (NOD SCID gamma) at initial implant, and following passages in NOD SCID (non-obese diabetic severe combined immunodeficiency) mice. Among 127 established PDX models from 441 surgically derived tumor specimens, all major histological subtypes were included: 52 adenocarcinomas, 62 squamous cell carcinomas, 1 adeno-squamous cell carcinomas, 5 sarcomatoid carcinomas, 5 large cell neuroendocrine carcinomas, and 2 small cell lung cancers. Over 100 PDX models have been profiled by next-generation exome sequencing (SureSelect Human 50Mbp kit), and array-based assays for copy number variant (HumanOmni 2.5-Quad BeadChip), DNA methylation (Infinium HumanMethylation450 BeadChip) and mRNA (DASL HumanHT-12 v4 BeadChip) profiles. Smaller subsets of PDXs have been characterized by mass spectrometry (MS)-based comprehensive proteome and protein-phosphotyrosine characterization. Genome/transcriptome/proteome profiles of 36 non-small cell lung carcinoma (NSCLC) PDX models correlated with patient primary tumors but to a much lesser extent with established NSCLC cell lines. A number of PDX models have genetic abnormalities linked to targeted therapies including mutations in EGFR (6), PIK3CA (13), and KRAS (21), and amplifications in FGFR1 (7) and CDK4 (6). This study provides the most solid evidence as yet that PDXs established from lung cancers mimic closely the genomic and proteomic characteristics of patient primary tumors and retain driver genetic abnormalities. Citation Format: Nhu-An Pham, Dennis Wang, Jiefei Tong, Chang-Qi Zhu, Lei Li, Wen Zhang, Ruoshi Shi, Shingo Sakashita, Melania Pintilie, Michael F. Moran, Geoffrey Liu, Ming-Sound Tsao. Fidelity of genomic and proteomic features of patient-derived xenografts of lung cancers. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B32.

Abstract B31: Establishment and molecular characterization of patient-derived tumor xenografts from resected tumors or ascites fluids of patients with pancreatic/ampullary/bile duct carcinomas

Pancreatic adenocarcinoma (PDAC) is the 4th most common cause of cancer deaths in North America, for both men and women with a 5-year survival rate of less than 5%. The poor prognosis rate is attributed to late presentation of the disease and the lack of effective treatment options. Large-scale genome sequencing efforts on PDAC tumors show evidence of high mutational burden and revealed a number of mutated genes affecting multiple oncogenic pathways. While there are significant endeavors in developing specific targeted agents against “driver” mutations, tumor diversity within and across patient population remains a key factor affecting therapeutic efficacy. In this context, the availability of large cohorts of genomically characterized patient-derived xenograft (PDX) tumor models may help to accelerate the development of novel therapies against this lethal cancer. PDX models provide a renewable resource to maintain a patient9s tumor ex vivo for pre-clinical or co-clinical studies. As part of The International Cancer Genome Consortium (ICGC), our laboratory has established 93 PDX models in non-obese diabetic and severe combined immune-deficient (NOD-SCID) mice from Whipple resection specimens. These tumors represent a heterogeneous group of neoplasms arising from the head, body and tail of pancreas, bile duct and Ampulla of Vater. All implantations including in the subcutaneous pocket at the flank or at the orthotopic pancreas site, were performed using 4-8 weeks old NOD-SCID mice. Successful growth and serial transplant to multiple mouse generations were observed in in 74 PDX models of the 93 implanted PDAC specimens, achieving an 80% engraftment rate, one of the highest reported in any type of cancer. Histology fidelity was preserved in the PDX models compared to corresponding patient tumors. Failed implants were due to specimens characterized by borderline malignancy and absence of tumor cells. Whole exome sequencing and copy number aberration profiling was completed for 61 PDXs and blood from the matched patients. Cancer-specific single nucleotide variation (SNV) load varied widely from 38 to 305 in PDXs. The most recurrent activating mutation was observed in KRAS with 77% of PDX models showing alterations at codon G12 (65%), G13 (8%) and Q61 (4%); in addition, 26% PDXs had a copy number gain in KRAS. Molecular comparisons of the 21 PDX models and their matched patient tumors showed that alternate allele frequency of KRAS mutation from exome sequencing of primary tumor is a strong indicator of the tumor cellularity; a higher tumor cellularity results in a larger overlap of cancer specific alterations between xenografts and corresponding patient tumors. We have demonstrated a successful establishment of PDX models that represent genomic architecture of major subclonal populations of patient PDAC primary tumors. Citation Format: Nikolina Radulovich, Emin Ibrahimov, Carson Holt, Vibha Raghavan, Tracy Zhao, Rob Denroch, Nhu-An Pham, Steve Gallinger, Melania Pintilie, Lincoln Stein, John McPherson, Lakshmi Muthuswamy, Ming Sound Tsao. Establishment and molecular characterization of patient-derived tumor xenografts from resected tumors or ascites fluids of patients with pancreatic/ampullary/bile duct carcinomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B31.