Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

Prevalence and Clinicopathologic Characteristics of the Molecular Subtypes in Malignant Glioma: A Multi-Institutional Analysis of 941 Cases

Background

Glioblastoma can be classified into four distinct molecular subtypes (Proneural, Neural, Classical and Mesenchymal), based on gene expression profiling. This study aimed to investigate the prevalence, clinicopathologic features and overall survival (OS) of the four molecular subtypes among all malignant gliomas.

Methods

A total of 941 gene expression arrays with clinical data were obtained from the Rembrandt, {"type":"entrez-geo","attrs":{"text":"GSE16011","term_id":"16011"}}GSE16011 and CGGA datasets. Molecular subtypes were predicted with a prediction analysis of microarray.

Results

Among 941 malignant gliomas, 32.73% were Proneural, 15.09% Neural, 19.77% Classical and 32.41% Mesenchymal. The Proneural and Neural subtypes occurred largely in low-grade gliomas, while the Classical and Mesenchymal subtypes were more frequent in high-grade gliomas. A survival analysis showed that the Proneural subtype displayed a good prognosis, Neural had an intermediate correlation with overall survival, Mesenchymal had a worse prognosis than Neural, and Classical had the worst clinical outcome. Furthermore, oligodendrocytomas were preferentially assigned to the Proneural subtype, while the Mesenchymal subtype included a higher percentage of astrocytomas, compared with oligodendrocytomas. Additionally, nearly all classical gliomas harbored EGFR amplifications. Classical anaplastic gliomas have similar clinical outcomes as their glioblastoma counterparts and should be treated more aggressively.

Conclusions

Molecular subtypes exist stably in all histological malignant gliomas subtypes. This could be an important improvement to histological diagnoses for both prognosis evaluations and clinical outcome predictions.     

Establishment and Characterization of Primary Glioblastoma Cell Lines from Fresh and Frozen Material: A Detailed Comparison

Background

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. High molecular heterogeneity of GBM tumors is well recognized, forming the rationale for molecular tests required before administration of several of the novel therapeutics rapidly entering the clinics. One model that has gained wide acceptance is the primary cell culture model. The laborious and time consuming process is rewarded with a relative high success rate (about 60%). We here describe and evaluate a very simple cryopreservation procedure for GBM tissue prior to model establishment that will considerably reduce the logistic complexity.

Methods

Twenty-seven GBM samples collected ad hoc were prepared for primary cell culture freshly from surgery (#1) and after cryopreservation (#2).

Results

Take rates after cryopreservation (59%) were as satisfactory as from fresh tissue (63%; p = 1.000). We did not observe any relevant molecular or phenotypic differences between cell lines established from fresh or vitally frozen tissue. Further, sensitivity both towards standard chemotherapeutic agents (Temozolomide, BCNU and Vincristine) and novel agents like the receptor tyrosine kinase inhibitor Imatinib did not differ.

Conclusions

Our simple cryopreservation procedure facilitates collection, long-time storage and propagation (modeling) of clinical GBM specimens (potentially also from distant centers) for basic research, (pre-) clinical studies of novel therapies and individual response prediction.     

Establishment of Human Patient-Derived Endometrial Cancer Xenografts in NOD scid Gamma Mice for the Study of Invasion and Metastasis

Objective

Most endometrial cancers are detected early and have a good prognosis, while some endometrial cancers are highly invasive, metastasize early, and respond suboptimally to therapy. Currently, appropriate model systems to study the aggressive nature of these tumors are lacking. The objective of this study was to establish a mouse xenograft model of endometrial tumors derived from patients in order to study the biological aggressive characteristics that underlie invasion and metastasis.

Methods

Endometrial tumor tissue fragments (1.5 mm×1.5 mm) from patients undergoing surgery, were transplanted under the renal capsule of NOD scid gamma mice. After 6–8 weeks, tumors were excised and serially transplanted into additional mice for propagation. Immunohistochemical analysis of the tumors was done for various tumor markers.

Results

Four cases of different subtypes of endometrial cancer were grown and propagated in mice. Three of the four tumor cases invaded into the kidneys and to adjacent organs. While all tumors exhibited minimal to no staining for estrogen receptor α, progesterone receptor staining was observed for tumor grafts. In addition, levels and localization of E-cadherin, cytokeratin and vimentin varied depending on subtype. Finally, all tumor xenografts stained positively for urokinase plasminogen activator while 3 tumor xenografts, which showed invasive characteristics, stained positively for urokinase plasminogen activator receptor.

Conclusion

Endometrial tumors transplanted under the renal capsule exhibit growth, invasion and local spread. These tumors can be propagated and used to study aggressive endometrial cancer.     

Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells

Background

Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells.

Methods

Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways.

Results

The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation.

Conclusions

Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.

     

Fasting Enhances the Response of Glioma to Chemo- and Radiotherapy

Background

Glioma, including anaplastic astrocytoma and glioblastoma multiforme (GBM) are among the most commonly diagnosed malignant adult brain tumors. GBM is a highly invasive and angiogenic tumor, resulting in a 12 to 15 months median survival. The treatment of GBM is multimodal and includes surgical resection, followed by adjuvant radio-and chemotherapy. We have previously reported that short-term starvation (STS) enhances the therapeutic index of chemo-treatments by differentially protecting normal cells against and/or sensitizing tumor cells to chemotoxicity.

Methodology and Principal Findings

To test the effect of starvation on glioma cells in vitro, we treated primary mouse glia, murine GL26, rat C6 and human U251, LN229 and A172 glioma cells with Temozolomide in ad lib and STS mimicking conditions. In vivo, mice with subcutaneous or intracranial models of GL26 glioma were starved for 48 hours prior to radio- or chemotherapy and the effects on tumor progression and survival were measured. Starvation-mimicking conditions sensitized murine, rat and human glioma cells, but not primary mixed glia, to chemotherapy. In vivo, starvation for 48 hours, which causes a significant reduction in blood glucose and circulating insulin-like growth factor 1 (IGF-1) levels, sensitized both subcutaneous and intracranial glioma models to radio-and chemotherapy.

Conclusion

Starvation-induced cancer sensitization to radio- or chemotherapy leads to extended survival in the in vivo glioma models tested. These results indicate that fasting and fasting-mimicking interventions could enhance the efficacy of existing cancer treatments against aggressive glioma in patients.     

Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials

Background

Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients.

Findings

Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs present in tumors, draining lymph nodes and spleen and improved long-term survival (50% of mice survived >150 days). No improvement in survival was observed when Tregs were depleted 24 days after tumor implantation, suggesting that tumor burden is an important factor for determining efficacy of Treg depletion in clinical trials. In a T cell dependent model of brain tumor regression elicited by intratumoral delivery of adenoviral vectors (Ad) expressing Fms-like Tyrosine Kinase 3 ligand (Flt3L) and Herpes Simplex Type 1-Thymidine Kinase (TK) with ganciclovir (GCV), we demonstrate that administration of PC61 24 days after tumor implantation (7 days after treatment) inhibited T cell dependent tumor regression and long term survival. Further, depletion with PC61 completely inhibited clonal expansion of tumor antigen-specific T lymphocytes in response to the treatment.

Conclusions

Our data demonstrate for the first time, that although Treg depletion inhibits the progression/eliminates GBM tumors, its efficacy is dependent on tumor burden. We conclude that this approach will be useful in a setting of minimal residual disease. Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity.     

Analysis of FoxP3+ T-Regulatory Cells and CD8+T-Cells in Ovarian Carcinoma: Location and Tumor Infiltration Patterns Are Key Prognostic Markers

Purpose

Tumor infiltrating CD4+CD25+FoxP3+ regulatory immune cells (Treg) have been associated with impaired anti- tumor immune response and unfavorable prognosis for patients affected by ovarian carcinoma, whereas CD8+ T-cells have been found to positively influence survival rates in a large panel of solid tumors. Recently, density, location and tumor infiltration patterns of the respective immune cell subtypes have been identified as key prognostic factors for different types of tumors.

Patients and Methods

We stained 210 human ovarian carcinoma samples immunhistochemically for FoxP3 and CD8 to identify the impact different immune cell patterns have on generally accepted prognostic variables as well as on overall survival.

Results

We found that FoxP3+ cells located within lymphoid aggregates surrounding the tumor were strongly associated with reduced survival time (P = 0.007). Central accumulation of CD8+ effector cells within the tumor bed shows a positive effect on survival (P = 0,001).

Conclusion

The distribution pattern of immune cells within the tumor environment strongly influences prognosis and overall survival time of patients with ovarian carcinoma.     

Genetic Instability and Intratumoral Heterogeneity in Neuroblastoma with MYCN Amplification Plus 11q Deletion

Background/Aim

Genetic analysis in neuroblastoma has identified the profound influence of MYCN amplification and 11q deletion in patients’ prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features.

Methods

We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques.

Results and Conclusion

This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.     

Tumor Development,Growth Characteristics and Spectrum of Genetic Aberrations in the TH-MYCN Mouse Model of Neuroblastoma

Background

The TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies.

Methods

We followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations.

Results

DNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6–19 weeks (median 9.1) and homozygous mice at 4.0–6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17.

Conclusion

Hemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.     

Clinical,Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens

Background

Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC.

Methods

Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression.

Results

Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines.

Conclusions

The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.     

Tumor characterization and stratification by integrated molecular profiles reveals essential pan-cancer features

Background

Identification of tumor heterogeneity and genomic similarities across different cancer types is essential to the design of effective stratified treatments and for the discovery of treatments that can be extended to different types of tumors. However, systematic investigations on comprehensive molecular profiles have not been fully explored to achieve this goal.

Results

Here, we performed a network-based integrative pan-cancer genomic analysis on >3000 samples from 12 cancer types to uncover novel stratifications among tumors. Our study not only revealed recurrently reported cross-cancer similarities, but also identified novel ones. The macro-scale stratification demonstrates strong clinical relevance and reveals consistent risk tendency among cancer types. The micro-scale stratification shows essential pan-cancer heterogeneity with subgroup-specific gene network characteristics and biological functions.

Conclusions

In summary, our comprehensive network-based pan-cancer stratification provides valuable information about inter- and intra- cancer stratification for patient clinical assessments and therapeutic strategies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1687-x) contains supplementary material, which is available to authorized users.     

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

Methodology/Principal Findings

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

Conclusions/Significance

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.     

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution

Background

Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.

Results

We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma.

Conclusions

In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0433-z) contains supplementary material, which is available to authorized users.     

Label-Free Delineation of Brain Tumors by Coherent Anti-Stokes Raman Scattering Microscopy in an Orthotopic Mouse Model and Human Glioblastoma

Background

Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors.

Methods

Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor''s localization, cell proliferation and vascularization.

Results

The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma.

Conclusions

CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.