Molecular heterogeneity of non‐small cell lung carcinoma patient‐derived xenografts closely reflect their primary tumors

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient‐derived tumor xenograft (PDX) resource from surgically resected non‐small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non‐obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non‐neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)‐proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno‐squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY‐proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.     

Vimentin DNA methylation predicts survival in breast cancer

The Vimentin gene plays a pivotal role in epithelial-to-mesenchymal transition and is known to be overexpressed in the prognostically poor basal-like breast cancer subtype. Recent studies have reported Vimentin DNA methylation in association with poor clinical outcomes in other solid tumors, but not in breast cancer. We therefore quantified Vimentin DNA methylation using MALDI-TOF mass spectrometry in breast tumors and matched normal pairs in association with gene expression and survival in a hospital-based study of breast cancer patients. Gene expression data via qRT-PCR in cell lines and oligomicroarray data from breast tissues were correlated with percent methylation in the Vimentin promoter. A threshold of 20 percent average methylation compared with matched normal pairs was set for bivariate and multivariate tests of association between methylation and tumor subtype, tumor histopathology, and survival. Vimentin was differentially methylated in luminal breast cancer cell lines, and in luminal A, luminal B, and HER2-enriched breast tumor subtypes, but was rare in basal-like cell lines and tumors. Increased methylation was strongly correlated with decreased mRNA expression in cell lines, and had a moderate inverse correlation in breast tumors. Vimentin methylation predicted poor overall survival independent of race, subtype, stage, nodal status, or metastatic disease and holds promise as a new prognostic biomarker for breast cancer patients.     

Cytogenomic profiling of breast cancer brain metastases reveals potential for repurposing targeted therapeutics

Breast cancer brain metastases remain a significant clinical problem. Chemotherapy is ineffective and a lack of treatment options result in poor patient outcomes. Targeted therapeutics have proven to be highly effective in primary breast cancer, but lack of molecular genomic characterization of metastatic brain tumors is hindering the development of new treatment regimens. Here we contribute to fill this void by reporting on gene copy number variation (CNV) in 10 breast cancer metastatic brain tumors, assayed by array comparative genomic hybridization (aCGH). Results were compared to a list of cancer genes verified by others to influence cancer. Cancer gene aberrations were identified in all specimens and pathway-level analysis was applied to aggregate data, which identified stem cell pluripotency pathway enrichment and highlighted recurring, significant amplification of SOX2, PIK3CA, NTRK1, GNAS, CTNNB1, and FGFR1. For a subset of the metastatic brain tumor samples (n=4) we compared patient-matched primary breast cancer specimens. The results of our CGH analysis and validation by alternative methods indicate that oncogenic signals driving growth of metastatic tumors exist in the original cancer. This report contributes support for more rapid development of new treatments of metastatic brain tumors, the use of genomic-based diagnostic tools and repurposed drug treatments.     

Patient-derived cell models as preclinical tools for genome-directed targeted therapy

Background

In this study, we established patient-derived tumor cell (PDC) models using tissues collected from patients with metastatic cancer and assessed whether these models could be used as a tool for genome-based cancer treatment.

Methods

PDCs were isolated and cultured from malignant effusions including ascites and pleural fluid. Pathological examination, immunohistochemical analysis, and genomic profiling were performed to compare the histological and genomic features of primary tumors, PDCs. An exploratory gene expression profiling assay was performed to further characterize PDCs.

Results

From January 2012 to May 2013, 176 samples from patients with metastatic cancer were collected. PDC models were successfully established in 130 (73.6%) samples. The median time from specimen collection to passage 1 (P1) was 3 weeks (range, 0.5–4 weeks), while that from P1 to P2 was 2.5 weeks (range, 0.5–5 weeks). Sixteen paired samples of genomic alterations were highly concordant between each primary tumor and progeny PDCs, with an average variant allele frequency (VAF) correlation of 0.878. We compared genomic profiles of the primary tumor (P0), P1 cells, P2 cells, and patient-derived xenografts (PDXs) derived from P2 cells and found that three samples (P0, P1, and P2 cells) were highly correlated (0.99–1.00). Moreover, PDXs showed more than 100 variants, with correlations of only 0.6–0.8 for the other samples. Drug responses of PDCs were reflective of the clinical response to targeted agents in selected patient PDC lines.

Conclusion(s)

Our results provided evidence that our PDC model was a promising model for preclinical experiments and closely resembled the patient tumor genome and clinical response.     

Ovarian carcinoma patient derived xenografts reproduce their tumor of origin and preserve an oligoclonal structure

Advanced Epithelial Ovarian Cancer (EOC) patients frequently relapse by 24 months and develop resistant disease. Research on EOC therapies relies on cancer cell lines established decades ago making Patient Derived Xenografts (PDX) attractive models, because they are faithful representations of the original tumor. We established 35 ovarian cancer PDXs resulting from the original graft of 77 EOC samples onto immuno-compromised mice. PDXs covered the diversity of EOC histotypes and graft take was correlated with early patient death. Fourteen PDXs were characterized at the genetic and histological levels. PDXs reproduced phenotypic features of the ovarian tumors of origin and conserved the principal characteristics of the original copy number change (CNC) profiles over several passages. However, CNC fluctuations in specific subregions comparing the original tumor and the PDXs indicated the oligoclonal nature of the original tumors. Detailed analysis by CGH, FISH and exome sequencing of one case, for which several tumor nodules were sampled and grafted, revealed that PDXs globally maintained an oligoclonal structure. No overgrowth of a particular subclone present in the original tumor was observed in the PDXs. This suggested that xenotransplantation of ovarian tumors and growth as PDX preserved at least in part the clonal diversity of the original tumor. We believe our data reinforce the potential of PDX as exquisite tools in pre-clinical assays.     

Prognostic significance of tissue transglutaminase in drug resistant and metastatic breast cancer.

PURPOSE: Drug resistance and metastasis pose major impediments in the successful treatment of cancer. We previously reported that multidrug-resistant breast cancer cells exhibit high levels of tissue transglutaminase (TG2; EC 2.3.2.13). Because the drug-resistant and metastatic phenotypes are thought to share some common pathways, we sought to determine whether metastatic breast cancer cells express high levels of TG2. EXPERIMENTAL DESIGN: The metastatic breast cancer cell line MDA-MB-231 and the sublines derived from it were tested for TG2 expression. Similarly, several sublines derived from an immortal but normal breast epithelial cell line, MCF10A, representing various stages in breast cancer progression were studied for TG2 expression. The primary and nodal tumor samples from 30 patients with breast cancer were also studied for TG2 expression. RESULTS: The MDA-MB-231 cells expressed high basal levels of TG2. Two clones derived from this cell line, MDA231/cl.9 and MDA231/cl.16, showed a 10- to 15-fold difference in TG2 level. TG2-deficient MDA231/cl.9 cells exhibited higher sensitivity to doxorubicin and were less invasive than were the TG2-sufficient MDA231/cl.16 cells. The MCF10A-derived sublines had increased TG2 expression as they advanced from noninvasive to an invasive phenotype. Importantly, the metastatic lymph node tumors from patients with breast cancer showed significant higher levels of TG2 expression compared with the primary tumors from the same patients. CONCLUSIONS: TG2 expression is up-regulated in drug-resistant and metastatic breast cancer cells, and it can serve as a valuable prognostic marker for these phenotypes.     

Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16^ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16^ink4a may indicate poor efficacy of CDK4 inhibitors.     

基于穿刺活检的复发转移性结直肠癌患者源性异种移植模型的建立

背景与目的:现行的结直肠癌患者源性异种移植(patient-derived xenografts,PDXs)模型建模样本多来源于外科手术,但复发转移性结直肠癌(metastatic colorectal cancer,mCRC)患者手术机会少,难以获取标本.该研究旨在利用穿刺活检术建立mCRC的PDXs模型.方法:入组12例结直肠癌根治术后患者,存在临床症状和(或)影像学提示术后复发和(或)转移,需行穿刺活检明确诊断、且不存在穿刺活检禁忌证者,保留用于常规病理诊断包括基因检测的组织量后,剩余的标本用于PDXs模型的建立.结果:共成功建立7例mCRC的PDXs模型,成功率为77.8%.结论:基于穿刺活检术建立mCRC的PDXs模型成功率高,可较完整的复制原始肿瘤特性,且操作安全、简便.     

Upregulation of DF3, in association with ICAM-1 and MHC class II by IFN-gamma in short-term human mammary carcinoma cell cultures

This study was designed to determine whether in vitro exposure of isolated short-term human primary and metastatic breast tumor cell cultures to interferon-gamma (IFN-gamma) could enhance expression of the breast tumor associated DF3 antigen in association with the intercellular adhesion molecule 1 (ICAM-1) and MHC class II molecules. Cell cultures were established from primary solid tumors and metastatic cells as previously described (Sgagias et al., 1995). Data show that recombinant human IFN-gamma treatment, in vitro, dramatically increased the breast tumor associated DF3 antigen, in association with ICAM-1, and MHC class II antigens in primary breast cancer cell cultures. All primary breast tumor cell cultures constitutively expressed high levels of HLA-class I antigen. Metastatic breast cancer cell cultures expressed high levels of DF3 and recombinant human IFN-gamma treatment, in vitro, upregulated ICAM-1 and MHC class II antigens before and after passage of the metastatic cells through the nude mouse. Metastatic breast cancer cells similar to primary breast cancer cells constitutively expressed high levels of MHC class I antigens. In addition, three LAK cell lines significantly lysed the primary and the metastatic breast tumor cell cultures to the same degree before and after passage of the metastatic cancer cells through the nude mouse. These data indicate the upregulation of the breast tumor associated DF3 antigen in vitro after IFN-gamma treatment and its persistence in vivo, after passage of the metastatic breast cancer cells through the nude mouse. The ability of IFN-gamma to upregulate the breast tumor associated DF3 antigen in association with the ICAM-1 and HLA class II antigens may play an important role in eliciting an immune response which may contribute to the immunodiagnosis, and immunotherapy of breast cancer.     

Establishment of novel cell lines recapitulating the genetic landscape of uveal melanoma and preclinical validation of mTOR as a therapeutic target

Uveal melanoma (UM) is the most common primary tumor of the eye in adults. There is no standard adjuvant treatment to prevent metastasis and no effective therapy in the metastatic setting. We have established a unique panel of 7 UM cell lines from either patient''s tumors or patient‐derived tumor xenografts (PDXs). This panel recapitulates the molecular landscape of the disease in terms of genetic alterations and mutations. All the cell lines display GNAQ or GNA11 activating mutations, and importantly four of them display BAP1 (BRCA1 associated protein‐1) deficiency, a hallmark of aggressive disease. The mTOR pathway was shown to be activated in most of the cell lines independent of AKT signaling. mTOR inhibitor Everolimus reduced the viability of UM cell lines and significantly delayed tumor growth in 4 PDXs. Our data suggest that mTOR inhibition with Everolimus, possibly in combination with other agents, may be considered as a therapeutic option for the management of uveal melanoma.     

The ELEANOR noncoding RNA expression contributes to cancer dormancy and predicts late recurrence of estrogen receptor‐positive breast cancer

The recurrence risk of estrogen receptor (ER)‐positive breast cancer remains high for a long period of time, unlike other types of cancer. Late recurrence reflects the ability of cancer cells to remain dormant through various events, including cancer stemness acquisition, but the detailed mechanism is unknown. ESR1 locus enhancing and activating noncoding RNAs (ELEANORS) are a cluster of nuclear noncoding RNAs originally identified in a recurrent breast cancer cell model. Although their functions as chromatin regulators in vitro are well characterized, their roles in vivo remain elusive. In this study, we evaluated the clinicopathologic features of ELEANORS, using primary and corresponding metastatic breast cancer tissues. The ELEANOR expression was restricted to ER‐positive cases and well‐correlated with the ER and progesterone receptor expression levels, especially at the metastatic sites. ELEANORS were detected in both primary and metastatic tumors (32% and 29%, respectively), and frequently in postmenopausal cases. Interestingly, after surgery, patients with ELEANOR‐positive primary tumors showed increased relapse rates after, but not within, 5 years. Multivariate analysis showed that ELEANORS are an independent recurrence risk factor. Consistently, analyses with cell lines, mouse xenografts, and patient tissues revealed that ELEANORS upregulate a breast cancer stemness gene, CD44, and maintain the cancer stem cell population, which could facilitate tumor dormancy. Our findings highlight a new role of nuclear long noncoding RNAs and their clinical potential as predictive biomarkers and therapeutic targets for late recurrence of ER‐positive breast cancer.     

Combining serial analysis of gene expression and array technologies to identify genes differentially expressed in breast cancer.

Several methods have been used recently to determine gene expression profiles of cell populations. Here we demonstrate the strength of combining two approaches, serial analysis of gene expression (SAGE) and DNA arrays, to help elucidate pathways in breast cancer progression by finding genes consistently expressed at different levels in primary breast cancers, metastatic breast cancers, and normal mammary epithelial cells. SAGE profiles of 21PT and 21MT, two well-characterized breast tumor cell lines, were compared with SAGE profiles of normal breast epithelial cells to identify differentially expressed genes. A subset of these candidates was then placed on an array and screened with clinical breast tumor samples to find genes and expressed sequence tags that are consistently expressed at different levels in diseased and normal tissues. In addition to finding the predicted overexpression of known breast cancer markers HER-2/neu and MUC-1, the powerful coupling of SAGE and DNA arrays resulted in the identification of genes and potential pathways not implicated previously in breast cancer. Moreover, these techniques also generated information about the differences and similarities of expression profiles in primary and metastatic breast tumors. Thus, combining SAGE and custom array technology allowed for the rapid identification and validation of the clinical relevance of many genes potentially involved in breast cancer progression. These differentially expressed genes may be useful as tumor markers and prognostic indicators and may be suitable targets for various forms of therapeutic intervention.     

The estrogen-regulated anterior gradient 2 (AGR2) protein in breast cancer: a potential drug target and biomarker

Initially discovered as an estrogen-responsive gene in breast cancer cell lines, anterior gradient 2 (AGR2) is a developmentally regulated gene belonging to the protein disulfide isomerase (PDI) gene family. Developmentally, AGR2 is expressed in the mammary gland in an estrogen-dependent manner, and AGR2 knockout and overexpression mouse models indicate that the gene promotes lobuloalveolar development by stimulating cell proliferation. Although AGR2 overexpression alone seems insufficient for breast tumorigenesis in mice, several lines of investigations suggest that AGR2 promotes breast tumorigenesis. Overexpression of AGR2 in several breast cancer cell lines increases cell survival in clonogenic assays and cell proliferation, whereas AGR2 loss of function leads to decreased cell cycle progression and cell death. In addition, AGR2 was shown to promote metastasis of breast epithelial cells in an in vivo metastasis assay. As a PDI, AGR2 is thought to be involved in the unfolded protein response that alleviates endoplasmic reticulum stress. Since cancer has to overcome proteotoxic stress due to excess protein production, AGR2 may be one of many pro-survival factors recruited to assist in protein folding or degradation or both. When AGR2 is secreted, it plays a role in cellular adhesion and dissemination of metastatic tumor cells. In breast cancer, AGR2 expression is associated with estrogen receptor (ER)-positive tumors; its overexpression is a predictor of poor prognosis. The AGR2 gene is directly targeted by ER-alpha, which is preferentially bound in tumors with poor outcome. Whereas aromatase inhibitor therapy decreases AGR2 expression, tamoxifen acts as an agonist of AGR2 expression in ER-positive tumors, perhaps contributing to tamoxifen resistance. AGR2 is also overexpressed in a subset of ER-negative tumors. Furthermore, AGR2 expression is associated with the dissemination of metastatic breast cancer cells and can be used as a marker to identify circulating tumor cells and metastatic cells in sentinel lymph nodes. In conclusion, AGR2 is a promising drug target in breast cancer and may serve as a useful prognostic indicator as well as a marker of breast cancer metastasis.     

Clonal expansion and linear genome evolution through breast cancer progression from pre-invasive stages to asynchronous metastasis

Evolution of the breast cancer genome from pre-invasive stages to asynchronous metastasis is complex and mostly unexplored, but highly demanded as it may provide novel markers for and mechanistic insights in cancer progression. The increasing use of personalized therapy of breast cancer necessitates knowledge of the degree of genomic concordance between different steps of malignant progression as primary tumors often are used as surrogates of systemic disease. Based on exome sequencing we performed copy number profiling and point mutation detection on successive steps of breast cancer progression from one breast cancer patient, including two different regions of Ductal Carcinoma In Situ (DCIS), primary tumor and an asynchronous metastasis. We identify a remarkable landscape of somatic mutations, retained throughout breast cancer progression and with new mutational events emerging at each step. Our data, contrary to the proposed model of early dissemination of metastatic cells and parallel progression of primary tumors and metastases, provide evidence of linear progression of breast cancer with relatively late dissemination from the primary tumor. The genomic discordance between the different stages of tumor evolution in this patient emphasizes the importance of molecular profiling of metastatic tissue directing molecularly targeted therapy at recurrence.     

Amplified centrosomes in breast cancer: a potential indicator of tumor aggressiveness

Molecular mechanisms leading to genomic instability and phenotypic variation during tumor development and progression are poorly understood. Such instability represents a major problem in the management of breast cancer because of its contribution to more aggressive phenotypes as well as chemoresistance. In this study we analyzed breast carcinomas and tumor-derived cell lines to determine the relationship between centrosome amplification and established prognostic factors. Our results show that centrosome amplification can arise independent of ER or p53 status and is a common feature of aneuploid breast tumors. Centrosome amplification is associated with mitotic spindle abnormalities in breast carcinomas and thus may contribute to genomic instability and the development of more aggressive phenotypes during tumor progression.     

High HSP90 expression is associated with decreased survival in breast cancer

The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.