Patient-matched Normal Tissue Research Articles

Population bias in somatic measurement of microsatellite instability status.

Microsatellite instability (MSI) is a key secondary effect of a defective DNA mismatch repair mechanism resulting in incorrectly replicated microsatellites in many malignant tumors. Historically, MSI detection has been performed by fragment analysis (FA) on a panel of representative genomic markers. More recently, using next‐generation sequencing (NGS) to analyze thousands of microsatellites has been shown to improve the robustness and sensitivity of MSI detection. However, NGS‐based MSI tests can be prone to population biases if NGS results are aligned to a reference genome instead of patient‐matched normal tissue. We observed an increased rate of false positives in patients of African ancestry with an NGS‐based diagnostic for MSI status utilizing 7317 microsatellite loci. We then minimized this bias by training a modified calling model that utilized 2011 microsatellite loci. With these adjustments 100% (95% CI: 89.1% to 100%) of African ancestry patients in an independent validation test were called correctly using the updated model. This poses not only a significant technical improvement but also has an important clinical impact on directing immune checkpoint inhibitor therapy.

Increased MTH1-specific 8-oxodGTPase activity is a hallmark of cancer in colon, lung and pancreatic tissue

Cellular homeostasis is dependent on a balance between DNA damage and DNA repair mechanisms. Cells are constantly assaulted by both exogenous and endogenous stimuli leading to high levels of reactive oxygen species (ROS) that cause oxidation of the nucleotide dGTP to 8-oxodGTP. If this base is incorporated into DNA and goes unrepaired, it can result in G > T transversions, leading to genomic DNA damage. MutT Homolog 1 (MTH1) is a nucleoside diphosphate X (Nudix) pyrophosphatase that can remove 8-oxodGTP from the nucleotide pool before it is incorporated into DNA by hydrolyzing it into 8-oxodGMP. MTH1 expression has been shown to be elevated in many cancer cells and is thought to be a survival mechanism by which a cancer cell can stave off the effects of high ROS that can result in cell senescence or death. It has recently become a target of interest in cancer because it is thought that inhibiting MTH1 can increase genotoxic damage and cytotoxicity. Determining the role of MTH1 in normal and cancer cells is confounded by an inability to reliably and directly measure its native enzymatic activity. We have used the chimeric ATP-releasing guanine-oxidized (ARGO) probe that combines 8-oxodGTP and ATP to measure MTH1 enzymatic activity in colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) along with patient-matched normal tissue. MTH1 8-oxodGTPase activity is significantly increased in tumors across all three tissue types, indicating that MTH1 is a marker of cancer. MTH1 activity measured by ARGO assay was compared to mRNA and protein expression measured by RT-qPCR and Western blot in the CRC tissue pairs, revealing a positive correlation between ARGO assay and Western blot, but little correlation with RT-qPCR in these samples. The adoption of the ARGO assay will help in establishing the level of MTH1 activity in model systems and in assessing the effects of MTH1 modulation in the treatment of cancer.

PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

Tumor-specific antibody drugs can serve as cancer therapy with minimal side effects. A humanized antibody, PRL3-zumab, specifically binds to an intracellular oncogenic phosphatase PRL3, which is frequently expressed in several cancers. Here we show that PRL3-zumab specifically inhibits PRL3+ cancer cells in vivo, but not in vitro. PRL3 antigens are detected on the cell surface and outer exosomal membranes, implying an ‘inside-out’ externalization of PRL3. PRL3-zumab binds to surface PRL3 in a manner consistent with that in classical antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular phagocytosis tumor elimination pathways, as PRL3-zumab requires an intact Fc region and host FcγII/III receptor engagement to recruit B cells, NK cells and macrophages to PRL3+ tumor microenvironments. PRL3 is overexpressed in 80.6% of 151 fresh-frozen tumor samples across 11 common cancers examined, but not in patient-matched normal tissues, thereby implicating PRL3 as a tumor-associated antigen. Targeting externalized PRL3 antigens with PRL3-zumab may represent a feasible approach for anti-tumor immunotherapy.

Interleukin-6 is potential target to de-couple checkpoint inhibitor-induced colitis from antitumor immunity.

2616 Background: A deep understanding of the immunobiology of checkpoint inhibitor (CPI) induced immune related toxicities, such as immune related enterocolitis (irEC), and how these compare to the immune signatures in tumors could lead to the development of strategies that de-couple autoimmunity from anti-tumor immunity. Methods: Total RNA from patient-matched irEC and normal colon FFPE tissue from patients [n = 12] receiving CPIs were profiled with the 770 gene NanoString nCounter PanCancer Immune Profiling Panel (NanoPCIP). The mean fold change in gene expression from normal vs. irEC inflamed colonic tissue and baseline vs. on-treatment tumor samples from patients responding or non-responding to ipilimumab based therapy were analyzed. C57BL/6 mice with B16.BL6 melanoma tumors were treated with systemic anti-IL-6 + anti-CTLA-4 vs. anti-CTLA4 alone vs. placebo and tumor size was measured. Results: In patients with irEC, the highest significantly upregulated differentially expressed gene (DEG) in inflamed colon tissue encoded for IL-6 (Fold change +24.1). None of the significant and highest upregulated DEGs in the colitis, including IL-6, were significantly upregulated in responding tumors. Interestingly, IL-6 was also the highest upregulated DEG in non-responding tumors numerically. When comparing mean fold changes across these analyses, the gene with the largest difference in upregulatation between colitis and responding tumors was IL-6; the other highest upregulated genes in colitis encoded for neutrophil and monocyte chemotactic molecules. In our mouse models, the addition of IL-6 blockade to anti-CTLA-4 therapy significantly improved tumor shrinkage compared to anti-CTLA-4 alone. Conclusions: Our data demonstrates that IL-6-mediated inflammation may be more prevalent in irEC and tumors not responding to CPIs than in tumors responding, and blocking IL-6 enhances CPI anti-melanoma activity. Targeting IL-6 may ameliorate irEC without hindering anti-tumor immunity.

Abstract P2-06-06: Targeting loss of isoenzyme diversity as a novel therapeutic strategy in breast cancer

Abstract Background: Several metabolic steps are mediated by distinct proteins or isoenzymes that catalyze the same reaction, providing redundancy of metabolic functions. Metabolic states are frequently altered in cancer to support survival and proliferation in hypoxic and otherwise hostile microenvironments, and metabolic re-wiring often involve loss of isoenzyme diversity. We hypothesize that targeting enzymes that have lost isoenzyme diversity in cancer, but not in normal cells, provides an opportunity to selectively target cancers. In this study, we assessed mRNA expression of all known human isoenzyme families in breast cancer and normal breast tissue and identified isoenzymes with loss of diversity within each breast cancer subtype. Methods: We obtained RNAseq data from cancer and patient-matched normal breast tissues from the TCGA (N=66 HR+, N=24 HER2+, and N=15 TNBC tumors). We retrieved annotated human isoenzyme families from the ENZYME nomenclature database. We compared expression in cancer and matched normal samples from the same patient to identify isoenzymes that had i) same or increased expression of the target isoenzyme in cancer vs normal and ii) reduced expression of the complementary isoenzymes in cancer. We developed five scores that capture various elements of these characteristics and prioritized candidates as targets based on clustering and their combined ranking based on the five scores. We validated overexpression of the candidate isoenzymes relative to other isoforms in breast cancer microarray data from ArrayExpress (E-GEOD-76250: 33 TNBC, and E-GEOD-70951: 30 TNBC, 108 HR+, 10 HER2+). Results: We identified 321 enzymes in the TCGA discovery cohort that correspond to 829 unique isoenzymes. Overall, 636, 483 and 429 isoenzymes were differentially expressed in HR+, HER2+ and TNBC cancers, respectively, compared to corresponding normal samples. Of these, 308 isoenzymes were differentially expressed relative to normal in all 3 subtypes. In all, 112 and 92, and 84 were selected as candidate isoenzyme therapeutic targets in HR+, HER2+ and TNBC, respectively. 23 isoenzymes prioritized in clustering step were further validated. Finally, 6 isoenzymes were validated in HR+ (ALDOA, GUSB, GYG1, MIF, P3H1, PCK2), 10 in HER2+ (ALDH1L2, ALDOA, GLYATL2, GUSB, GYG1, GYS1, MIF, P3H1, PCK2, PTGS1) and 12 in TNBC (ADSS, ALAS1, ALDH1L2, ALDOA, ART3, GLYATL2, GUSB, GYS1, HS3ST1, MIF, PCK2, SOAT1), as potential targets for breast cancer treatment. Of these, 5 potential isoenzyme targets (ALDH1L2, GUSB, GLYATL2, MIF, PCK2), which were mostly hydrolases and transferases, were further selected for ongoing experimental validation in the laboratory. Decreased expression of the complementary isoforms of these 5 targets were primarily due to DNA methylation of the genes in cancer. Conclusions: We found that loss of isoenzyme diversity is a broad phenomenon in breast cancers that may be explored therapeutically. We identified several instances of “isoenzyme addiction” in which cancers depend exclusively on a single isoenzyme while downregulating via methylation the complementary isoenzymes, providing cancer-specific targeting opportunities. We are currently validating several of these targets in cell line models. Citation Format: Marczyk M, Gunasekharan V, Wali VB, Shi W, Patwardhan G, Qing T, Pusztai L, Hatzis C. Targeting loss of isoenzyme diversity as a novel therapeutic strategy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-06.

RNA based individualized drug selection in breast cancer patients without patient-matched normal tissue.

BackgroundWhile standard RNA expression tests stratify patients into risk groups, RNA-Seq can guide personalized drug selection based on expressed mutations, fusion genes, and differential expression (DE) between tumor and normal tissue. However, patient-matched normal tissue may be unavailable. Additionally, biological variability in normal tissue and technological biases may confound results. Therefore, we present normal expression reference data for two sequencing methods that are suitable for breast biopsies.ResultsWe identified breast cancer related and drug related genes that are expressed uniformly across our normal samples. Large subsets of these genes are identical for formalin fixed paraffin embedded samples and fresh frozen samples. Adipocyte signatures were detected in frozen compared to formalin samples, prepared by surgeons and pathologists, respectively. Gene expression confounded by adipocytes was identified using fat tissue samples. Finally, immune repertoire statistics were obtained for healthy breast, tumor and fat tissues.ConclusionsOur reference data can be used with patient tumor samples that are asservated and sequenced with a matching aforementioned method. Coefficients of variation are given for normal gene expression. Thus, potential drug selection can be based on confidently overexpressed genes and immune repertoire statistics.Materials and MethodsNormal expression from formalin and frozen healthy breast tissue samples using Roche Kapa RiboErase (total RNA) (19 formalin, 9 frozen) and Illumina TruSeq RNA Access (targeted RNA-Seq, aka TruSeq RNA Exome) (11 formalin, 1 frozen), and fat tissue (6 frozen Access). Tumor DE using 10 formalin total RNA tumor samples and 1 frozen targeted RNA tumor sample.

Biomolecular analysis of matrix proteoglycans as biomarkers in non small cell lung cancer.

Matrix proteoglycans (PGs) have shown promise as biomarker in malignancies. We employed agarose gel eletrophoresis, quantitative real- time reverse transcription-polymerase chain reaction and immunohistochemistry to evaluate the content of sulfated glicosaminoglycans (chondroitin sulfate and heparan sulfate) and expression of PG (biglycan, glypican, perlecan, syndecan e versican) in patient-matched normal and tumor tissues obtained from resected specimens of lung cancer. A significant increase of heparan sulfate (HS) and chondroitin sulfate (CS) concentrations was found in tumor tissue samples when compared to normal lung tissue samples. HS was also significantly increased in adenocarcinomas compared to squamous cell carcinomas. PG gene expression, with exception of syndecan, were significantly decreased in tumor tissue compared to normal lung, coinciding with significant decrease of PG protein levels in tumor cells and stroma compared to normal lung tissue (Kappa coefficient 0.41, 0.42 and 0,28, respectively). Women patients (p = 0.02), non smokers (p = 0.05), T stage (p = 0.009), N stage (p = 0.03) and adenocarcinoma (p = 0.05) were associated with improved overall survival (OS). Patients presenting tumors with low concentration of sulfated GAG and high PGs levels presented better OS compared to patients with high concentration of sulfated GAG and low expression of PGs. Cox regression model controlled by gender, tobacco history and histological type, showed that patients with high perlecan and versican expression in tumor presented respectively high probability of life (β risk 11.64; 1.27 to 15.90) and low risk of death (β risk 0.11; 0.02-0.51). The combined approach suggest matrix (PGs) as biomarkers in lung cancer.

Glycosaminoglycans and glycolipids as potential biomarkers in lung cancer.

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

Abstract 2208: Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer

Abstract Objective: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Method: A recently described isolation method, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). The authors showed comparable results for their method (abbreviated HSP-EV here) and UC-EV, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium. Subsequently we applied this technique on secretomes of fresh human colorectal cancer (CRC) (n=17) and colon adenoma (n=4) tissue as well as patient-matched normal colon tissue. Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs. Conclusions: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting. Citation Format: Meike De Wit, Jaco Knol, Inge de Reus, Tim Tim Schelfhorst, Logan Bishop-Currey, Nicole van Grieken, Sander Piersma, Thang V. Pham, Remond J. Fijneman, Gerrit A. Meijer, Henk Verheul, Connie R. Jimenez. Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2017-2208

MA02.02 A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC

MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumor biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumors, including lung cancer. MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7th generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumor tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x103 cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumor volume was measured over time and harvested once the tumor mass measured 500mm3 and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumors and cisplatin resistance was investigated relative to cisplatin sensitive controls. Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT. A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.

Abstract 3885: Peptide-mediated ‘miniprep’ isolation of extracellular vesicles for high-throughput proteomics; method evaluation and application in colon cancer

Abstract Background: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Recently a novel isolation method was described, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). Using cancer secretome and biofluid samples, the authors showed enrichment of exosome markers for their method (abbreviated HSP-EV here) but a detailed description of the captured EV proteomes in comparison to the gold-standard ultracentrifugation (UC) method and application to small tumor proximal fluid samples is lacking. Approach: Here we used label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium to compare EV fractions captured using the new HSP peptide method and UC. Subsequently we applied this novel method to profile EVs released from fresh human colorectal tumors (CRC) (n = 17) and colon adenoma (n = 4) tissue as well as patient-matched normal colon tissue. Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs. Conclusion: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting. Citation Format: Connie R. Jimenez, Meike de Wit, Jaco C. Knol, Inge de Reus, Tim Schelfhorst, Logan Bishop-Currey, Valerie Dusseldorp, Nicole van Grieken, Robin Beekhof, Sander R. Piersma, Thang V. Pham, Egbert F. Smit, Remond JA Fijneman, Gerrit Meijer, Henk MW Verheul. Peptide-mediated ‘miniprep’ isolation of extracellular vesicles for high-throughput proteomics; method evaluation and application in colon 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 3885.

PRL3-zumab, a first-in-class humanized antibody for cancer therapy.

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3+, but not PRL-3-, orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3+ tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become "extracellular oncotargets" that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery.

Oxalate induces breast cancer.

BackgroundMicrocalcifications can be the early and only presenting sign of breast cancer. One shared characteristic of breast cancer is the appearance of mammographic mammary microcalcifications that can routinely be used to detect breast cancer in its initial stages, which is of key importance due to the possibility that early detection allows the application of more conservative therapies for a better patient outcome. The mechanism by which mammary microcalcifications are formed is still largely unknown but breast cancers presenting microcalcifications are more often associated with a poorer prognosis.MethodsWe combined Capillary Electrochromatography, histology, and gene expression (qRT-PCR) to analyze patient-matched normal breast tissue vs. breast tumor. Potential carcinogenicity of oxalate was tested by its inoculation into mice. All data were subjected to statistical analysis.ResultsTo study the biological significance of oxalates within the breast tumor microenvironment, we measured oxalate concentration in both human breast tumor tissues and adjoining non-pathological breast tissues. We found that all tested breast tumor tissues contain a higher concentration of oxalates than their counterpart non-pathological breast tissue. Moreover, it was established that oxalate induces proliferation of breast cells and stimulates the expression of a pro-tumorigenic gene c-fos. Furthermore, oxalate generates highly malignant and undifferentiated tumors when it was injected into the mammary fatpad in female mice, but not when injected into their back, indicating that oxalate does not induce cancer formation in all types of tissues. Moreover, neither human kidney-epithelial cells nor mouse fibroblast cells proliferate when are treated with oxalate.ConclusionsWe found that the chronic exposure of breast epithelial cells to oxalate promotes the transformation of breast cells from normal to tumor cells, inducing the expression of a proto-oncogen as c-fos and proliferation in breast cancer cells. Furthermore, oxalate has a carcinogenic effect when injected into the mammary fatpad in mice, generating highly malignant and undifferentiated tumors with the characteristics of fibrosarcomas of the breast. As oxalates seem to promote these differences, it is expected that a significant reduction in the incidence of breast cancer tumors could be reached if it were possible to control oxalate production or its carcinogenic activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1747-2) contains supplementary material, which is available to authorized users.

Virulence genes are a signature of the microbiome in the colorectal tumor microenvironment.

BackgroundThe human gut microbiome is associated with the development of colon cancer, and recent studies have found changes in the microbiome in cancer patients compared to healthy controls. Studying the microbial communities in the tumor microenvironment may shed light on the role of host–bacteria interactions in colorectal cancer. Here, we highlight the major shifts in the colorectal tumor microbiome relative to that of matched normal colon tissue from the same individual, allowing us to survey the microbial communities in the tumor microenvironment and providing intrinsic control for environmental and host genetic effects on the microbiome.MethodsWe sequenced the microbiome in 44 primary tumor and 44 patient-matched normal colon tissue samples to determine differentially abundant microbial taxa These data were also used to functionally characterize the microbiome of the cancer and normal sample pairs and identify functional pathways enriched in the tumor-associated microbiota.ResultsWe find that tumors harbor distinct microbial communities compared to nearby healthy tissue. Our results show increased microbial diversity in the tumor microenvironment, with changes in the abundances of commensal and pathogenic bacterial taxa, including Fusobacterium and Providencia. While Fusobacterium has previously been implicated in colorectal cancer, Providencia is a novel tumor-associated agent which has not been identified in previous studies. Additionally, we identified a clear, significant enrichment of predicted virulence-associated genes in the colorectal cancer microenvironment, likely dependent upon the genomes of Fusobacterium and Providencia.ConclusionsThis work identifies bacterial taxa significantly correlated with colorectal cancer, including a novel finding of an elevated abundance of Providencia in the tumor microenvironment. We also describe the predicted metabolic pathways and enzymes differentially present in the tumor-associated microbiome, and show an enrichment of virulence-associated bacterial genes in the tumor microenvironment. This predicted virulence enrichment supports the hypothesis that the microbiome plays an active role in colorectal cancer development and/or progression. Our results provide a starting point for future prognostic and therapeutic research with the potential to improve patient outcomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0177-8) contains supplementary material, which is available to authorized users.

Metabolic signatures of renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is characterized by the constitutive up-regulation of the hypoxia inducible factor-1. One of its target enzymes, pyruvate dehydrogenase (PDH) kinase 1 (PDHK1) showed increased protein expression in tumor as compared to patient-matched normal tissues. PDHK1 phosphorylated and inhibited PDH whose enzymatic activity was severely diminished, depriving the TCA cycle of acetylCoA. We and others have shown a decrease in the protein expressions of all respiratory complexes alluding to a compromise in oxidative phosphorylation (OXPHOS). On the contrary, we found that key parameters of OXPHOS, namely ATP biosynthesis and membrane potential were consistently measurable in mitochondria isolated from ccRCC tumor tissues. Interestingly, an endogenous mitochondrial membrane potential (MMP) was evident when ADP was added to mitochondria isolated from ccRCC but not in normal tissues. In addition, the MMP elicited in the presence of ADP by respiratory substrates namely malate/glutamate, succinate, α-ketoglutarate and isocitrate was invariably higher in ccRCC. Two additional hallmarks of ccRCC include a loss of uncoupling protein (UCP)-2 and an increase in UCP-3. Based on our data, we proposed that inhibition of UCP3 by ADP could contribute to the endogenous MMP observed in ccRCC and other cancer cells.

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

BackgroundGlycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.MethodsThe expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.ResultsExpression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.ConclusionNSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.

Abstract 4285: Structural variant breakpoint detection in advanced colorectal cancer

Abstract Introduction Development of colorectal cancer (CRC) is accompanied by genomic alterations that drive tumor initiation and progression. Gains and losses of large chromosome segments result in DNA copy number alterations and subsequently quantitative changes in mRNA and protein expression levels. Interestingly, the accompanying chromosome breakpoints represent structural variants (SV) that may affect gene architecture and thereby normal gene function. Aim The aim of this study was to identify recurrent SV breakpoints in advanced CRC. Methods Previously a series of 352 advanced CRC samples from CAIRO and CAIRO2 clinical studies [Koopman et al. Lancet 2007; Tol et al. N Engl J Med 2009] was characterized for genome-wide DNA copy number alterations. DNA from formalin-fixed paraffin-embedded (FFPE) tumor and patient-matched normal tissue was subjected to high-resolution array-Comparative Genomic Hybridization (Agilent 180K arrays). Using these data, we now determined the prevalence of recurrent breakpoints in genes in CRC by computational analysis. In addition, multiplexed amplicon analysis involving 48 cancer-related genes (Illumina TruSeq Amplicon Cancer Panel) was applied to determine mutation frequencies. Multi-Dendrix was used to identify modules of (mutually exclusively mutated) cancer driver genes. Results We identified 748 recurrent SV breakpoints in genes (FDR<0.1). The highest frequency of recurrent breakpoints was detected in MACROD2, in up to ∼40% of CRC samples. Most recurrent breakpoints occurred in less than 5% of all tumors, and have not been reported before in CRC. Mutation frequencies in APC, TP53 and KRAS were conform expectations (60%, 58%, and 48%, respectively). Multi-Dendrix analysis revealed modules of cancer driver genes that included both the commonly mutated CRC cancer genes as well as genes with recurrent breakpoints, suggesting that several of the SV breakpoint genes are candidate drivers of the carcinogenic process. Conclusions We were able to pinpoint the prevalence of 748 recurrent SV breakpoint regions in genes using array-CGH data from 352 CRC samples. Moreover, our studies revealed several breakpoints in genes that are mutually exclusive with the commonly mutated APC, KRAS, and TP53 genes, and therefore represent novel candidate cancer driver genes. Further studies are required to investigate their functional and clinical significance. Citation Format: E van den Broek, O Krijgsman, D Sie, JC Haan, M Komor, J Traets, DAM Heideman, MA van de Wiel, ID Nagtegaal, CJA Punt, B Carvalho, B Ylstra, GA Meijer, RJA Fijneman. Structural variant breakpoint detection in advanced colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4285. doi:10.1158/1538-7445.AM2014-4285

Tumor cells are dislodged into the pulmonary vein during lobectomy

Intraoperative tumor shedding may facilitate tumor dissemination. In earlier studies, shed tumor cells were defined primarily by cytomorphological examination, and normal epithelial cells could not always be distinguished from tumor cells. We sought to accurately identify tumor cells using single-cell sequencing and determine whether these cells were mobilized into the circulation during pulmonary lobectomy. Forty-two blood samples collected from the tumor-draining pulmonary vein at the end of lobectomy procedures were analyzed. Arrays of nanowells were used to enumerate and retrieve single EpCAM(+) cells. Targeted sequencing of 10 to 15 cells and nested polymerase chain reaction of single cells detected somatic mutations in shed epithelial cells consistent with patient-matched tumor but not normal tissue. The mean number of EpCAM(+) cells in video-assisted thoracoscopy (VATS) lobectomy (no wedge) specimens (n = 16) was 165 (median, 115; range, 0-509) but sampling cells from 3 patients indicated that only 0% to 38% of the EpCAM(+) cells were tumor cells. The mean number of EpCAM(+) cells in VATS lobectomy (wedge) specimens (n = 12) was 1128 (median, 197; range, 47-9406) and all of the EpCAM(+) cells were normal epithelial cells in 2 patients sampled. The mean number of EpCAM(+) cells in thoracotomy specimens (n = 14) was 238 (median, 22; range, 9-2920) and 0% to 50% of total EpCAM(+) cells were tumor cells based on 4 patients sampled. Surgery mobilizes tumor cells into the pulmonary vein, along with many normal epithelial cells. EpCAM alone cannot differentiate between normal and tumor cells. On the other hand, single-cell genetic approaches with patient-matched normal and tumor tissues can accurately quantify the number of shed tumor cells.

Carbohydrate-Containing Molecules as Potential Biomarkers in Colon Cancer

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

ANKS1B is a smoking-related molecular alteration in clear cell renal cell carcinoma

BackgroundAn association between cigarette smoking and increased risk of clear cell renal cell carcinoma (ccRCC) has been established; however, there are limited data regarding the molecular mechanisms that underlie this association. We used a multi-stage design to identify and validate genes that are associated with smoking-related ccRCC.MethodsWe first conducted a microarray study to compare gene expression patterns in patient-matched ccRCC and normal kidney tissues between patients with (n = 23) and without (n = 42) a history of smoking. Analyses were first stratified on obesity status (the other primary risk factor for ccRCC) and then combined and analyzed together. To identify genes where the fold change in smokers relative to non-smokers was different in tumor tissues in comparison to patient-matched normal kidney tissues, we identified Affymetrix probesets that had a significant tissue type-by-smoking status interaction pvalue. We then performed RT-PCR validation on the top eight candidate genes in an independent sample of 28 smokers and 54 non-smokers.ResultsWe identified 15 probesets that mapped to eight genes that had candidate associations with smoking-related ccRCC: ANKS1B, ACOT6, PPWD1, EYS, LIMCH1, CHRNA6, MT1G, and ZNF600. Using RT-PCR, we validated that expression of ANKS1B is preferentially down-regulated in smoking-related ccRCC.ConclusionWe provide the first evidence that ANKS1B expression is down regulated in ccRCC tumors relative to patient-matched normal kidney tissue in smokers. Thus, ANKS1B should be explored further as a novel avenue for early detection as well as prevention of ccRCC in smokers.