Distant Normal Tissues Research Articles

Abstract 146: DNA methylation age of paired tumor-normal breast tissue in Chinese women with breast cancer

Abstract Background: Epigenetic age, captured by DNA methylation changes, is thought to be more informative with respect to disease risk and progression, than chronological age. A few studies have associated epigenetic age acceleration (EAA), the difference between DNA methylation age (DNAm age) and chronological age, with BC risk and subtypes. However, most of these studies were conducted in Western populations. In this study, we examined EAA in an Asian population, in which BC incidence rate is lower and age at BC onset is younger compared to most Western populations. Methods: We performed genome-wide DNA methylation profiling of 97 tumor and 89 paired distant normal tissue samples collected from BC patients in Hong Kong (HK) using an Illumina MethylationEPIC array. Two independent datasets were used to compare results: The Cancer Genome Atlas (TCGA, n=525 tumor and n=88 adjacent normal) and healthy women from the Komen tissue bank (n=59). DNAm age was calculated using Horvath's model based on 353 CpGs. The significance of EAA was tested using a simple linear regression model. We also used a multivariate regression model to test for equality of slopes (EAA rates) among different BC subtypes or datasets, considering interaction terms between age and subtype/dataset. Results: The average age at BC diagnosis was 58 years old (range: 33-81) and the distribution of the molecular subtype based on PAM50 was 43.0%, 29.1%, and 27.9% for luminal A, luminal B, and HER2-enriched/basal tumors, respectively. As expected, DNAm age showed a stronger correlation with chronological age in normal tissue (r=0.78, p<.0001) than in tumor tissue (r=0.27, p=0.0075). The average EAAs in normal and tumor samples were 9.43 and 6.02 years, respectively. Among different BC subtypes, EAA in normal tissue did not vary by subtypes. However, in tumor samples, luminal patients showed positive EAA (average 10/13 years in luminal A/B, respectively), while HER2-enriched/basal patients showed a negative EAA (average -8 years), although the rate of EAA did not vary significantly by subtype. Analyses on TCGA data produced consistent results. When comparing the rate of DNAm acceleration in normal tissue of HK, TCGA, and Komen, HK patients had a significantly different rate (βHK=0.46) compared to TCGA (βTCGA=0.65, p=0.001) and Komen (βKomen=0.80, p<.0001). Conclusion: Consistent with previous studies, we found that EAA in tumor samples varied across tumor subtypes. We also found that HK BC patients' epigenetic age accelerated at a different rate compared to predominantly white TCGA and Komen women, suggesting a potential racial biological difference. Large studies in other Asian populations are warranted to confirm our findings, which may provide biological insight into racial heterogeneity of BC, especially with regard to age at onset. Citation Format: Hela Koka, Bin Zhu, Shelly Lap Ah Tse, Difei Wang, Maeve Kiely, Jennifer Lyn Guida, Priscilla Lee, Feng Wang, Cherry Wu, Koon Ho Tsang, Wing-cheong Chan, Sze Hong Law, Eric Karlins, Bin Zhu, Amy Hutchinson, Belynda Hicks, Xiaohong R. Yang. DNA methylation age of paired tumor-normal breast tissue in Chinese women with breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 146.

Clinical significance of HDAC1, -2 and -3 expression levels in esophageal squamous cell carcinoma.

The present study analyzed the expression of the histone deacetylase (HDAC) 1, 2 and 3 in primary esophageal squamous cell carcinoma (ESCC) samples and how their levels correlate with clinicopathological parameters. ESCC patients (n=88) in the present study had received no previous treatment before undergoing surgical excision. The mRNA expression of HDAC1, -2 and -3 were detected by semi-quantified PCR in ESCC samples and distal normal samples. The relationship of HDAC1, -2 and -3 expression with clinicopathological parameters was analyzed by χ2 test. The correlation among these HDACs was analyzed by Pearson's correlation test. Compared with distal normal tissues, ESCC samples had higher expression of HDAC1, but not HDAC2 or HDAC3 (P<0.05). The expression of HDACs was different between Kazak and Han ethnicities. The expression of HDAC2 was correlated with invasion depth (P<0.05), but not with sex, age, metastasis, or the degree of tumor differentiation (P>0.05). There was no association between HDAC1 or HDAC3 and clinicopathological parameters (P>0.05). For the Kazak and Han ethnicities, HDAC1 expression was present in male patients, patients with well/moderate differentiated ESCC and T3 and T4 ESCC (P<0.01). HDAC1 in patients aged <60 was associated with ethnicity (P<0.05). HDAC2 expression was different in positive LN metastasis, well/moderate differentiation and T3 and T4 ESCC (P<0.01). HDAC3 expression in male patients, patients with negative LN metastasis and well/moderate differentiation ESCC was associated with ethnicity (P<0.05). Additionally, the expression levels of HDAC1, -2 and -3 did not correlate with each other. Thus, HDAC1 expression may be used as a risk factor for ESCC and HDAC2 levels may be used to predict invasion depth. The expression of HDAC1, -2 and -3 has ethnic differences.

Landscape of transcriptome variations uncovering known and novel driver events in colorectal carcinoma

We focused on an integrated view of genomic changes in Colorectal cancer (CRC) and distant normal colon tissue (NTC) to test the effectiveness of expression profiling on identification of molecular targets. We performed transcriptome on 16 primary coupled CRC and NTC tissues. We identified pathways and networks related to pathophysiology of CRC and selected potential therapeutic targets. CRC cells have multiple ways to reprogram its transcriptome: a functional enrichment analysis in 285 genes, 25% mutated, showed that they control the major cellular processes known to promote tumorigenesis. Among the genes showing alternative splicing, cell cycle related genes were upregulated (CCND1, CDC25B, MCM2, MCM3), while genes involved in fatty acid metabolism (ACAAA2, ACADS, ACAT1, ACOX, CPT1A, HMGCS2) were downregulated. Overall 148 genes showed differential splicing identifying 17 new isoforms. Most of them are involved in the pathogenesis of CRC, although the functions of these variants remain unknown. We identified 2 in-frame fusion events, KRT19-KRT18 and EEF1A1-HSP90AB1, encoding for chemical proteins in two CRC patients. We draw a functional interactome map involving integrated multiple genomic features in CRC. Finally, we underline that two functional cell programs are prevalently deregulated and absolutely crucial to determinate and sustain CRC phenotype.

Cyclin-dependent kinase 6 (CDK6) is a candidate diagnostic biomarker for early non-small cell lung cancer

BackgroundCyclin-dependent kinase 6 (CDK6) is an important regulatory protein of the cell cycle and plays an important role in tumor progression. The aim of this study was to investigate the expression of CDK6 in T1 stage non-small cell lung cancer (NSCLC) and to explore the association of CDK6 with the clinicopathological features of the disease.MethodsCDK6 expression was analyzed by real-time PCR (RT-PCR) and immunohistochemistry (IHC) in tumor tissue samples and the distal normal tissue samples from 56 T1 stage NSCLC patients. The correlation between CDK6 expression and clinicopathological features was analyzed using the independent samples t-test and nonparametric tests.ResultsWe found CDK6 had a tendency to increase in tumor tissues compared to normal tissues at the transcriptional level (P=0.073). Moreover, the expression of CDK6 protein in NSCLC tissues was also significantly higher than in normal lung tissues (P=0.003). With an increase of smoking quantity, the expression of CDK6 mRNA was increased (P=0.009). Remarkably, CDK6 expression was increased in squamous cell carcinoma (SCC) tissues but decreased in adenocarcinoma (AD) tissues at both the transcription and protein levels (P<0.001). After stratification based on pathological type, CDK6 gene expression was not associated with any clinicopathological features in SCC, while it was negatively associated with tumor diameter in AD (P=0.049).ConclusionsTaken together, these results indicated that abnormal expression of the CDK6 gene in NSCLC might be associated with pathological type, which may serve as a diagnostic biomarker for NSCLC.

Altered 5-Hydroxymethylcytosine Landscape in Primary Gastric Adenocarcinoma.

Multiple factors, including genetic and epigenetic fluctuations, have been linked to gastric cancer formation and progression. Cytosine methylation (5mC) has been recognized as a critical epigenetic mark in the mammalian genome. The recent discovery of 5-hydroxymethylcytosine (5hmC), which is generated by ten-eleven translocation (TET) enzymes, provides new perspectives to understand DNA methylation-related plasticity. In this study, we show that gastric tumors display significant loss of 5hmC. Using matched distant normal, peripheral, and tumor primary tissues, we performed genome-wide profiling of 5hmC and identified differentially hydroxymethylated regions (DhMRs) specifically associated with gastric tumors. Gene ontology analyses indicated that DhMRs (both loss of 5hmC and gain of 5hmC) were enriched among the genes involved in specific pathways. Interestingly, the binding motif of hypoxia-inducible factor 1 (HIF1) is enriched among both peripheral and tumor DhMRs, while the Myc-binding motif is specifically enriched among only tumor DhMRs. Tumor progression analyses revealed a unique set of DhMRs that correlate with tumor progression. These data together suggest that alteration of 5hmC could potentially contribute to the tumorigenesis of gastric tumors.

Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma.

Eukaryotic initiation factor 3c (eIF3C) is involved in the initiation of protein translation. Aberrant eIF3C expression has been reported in different types of human cancer. The present study aimed to assess the role of eIF3C in the malignant behavior of renal cell carcinoma invitro and invivo. eIF3C expression was assessed in 16 pairs of renal cell carcinoma (RCC) and matched distant normal tissues, and in RCC cell lines using immunohistochemistry. Subsequently, eIF3C was depleted using lentiviral short hairpin RNA and cell proliferation, cell cycle distribution and apoptosis of these eIF3C‑depleted cells were examined. Additionally, tumor cell xenograft assays in nude mice, Affymetrix microarrays and ingenuity pathway analyses were performed. eIF3C expression was upregulated in RCC tissues and cell lines. Depletion of eIF3C reduced tumor cell proliferation and arrested them at the G1 stage, thus promoting their apoptosis invitro. Depletion of eIF3C also inhibited the formation and growth of tumor cell xenografts in nude mice. In addition, depletion of eIF3C altered the expression levels of 994 differentially expressed genes in RCC cells (516 genes were upregulated and 478genes were downregulated). The expression levels of phosphorylated‑AKT, c‑JUN and NFKB inhibitor α were lower in the shorth hairpin RNA eIF3C‑transfected RCC cells compared with in the control group. In conclusion, the present study demonstrated that upregulated eIF3C expression contributed to the development and progression of RCC. Future studies should further evaluate whether eIF3C could be used as a potential strategy for RCC targeting therapy.

FRK plays an oncogenic role in non-small cell lung cancer by enhancing the stemness phenotype via induction of metabolic reprogramming.

The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.

Telomere Maintenance-Associated PML Is a Potential Specific Therapeutic Target of Human Colorectal Cancer

Telomere length maintenance is essential for cell proliferation, which is particularly prominent in cancer. We validate that the primary colorectal tumors exhibit heterogeneous telomere lengths but mostly (90%) short telomeres relative to normal tissues. Intriguingly, relatively short telomeres are associated with tumor malignancy as indicated by poorly differentiated state, and these tumors contain more cancer stem-like cells (CSLCs) identified by several commonly used markers CD44, EPHB2 or LGR5. Moreover, promyelocytic leukemia (PML) and ALT-associated PML nuclear bodies (APBs) are frequently found in tumors with short telomeres and high proliferation. In contrast, distant normal tissues rarely or only minimally express PML. Inhibition of PML and APBs by an ATR inhibitor decreases proliferation of CSLCs and organoids, suggesting a potential therapeutic target to progressive colorectal tumors. Together, telomere maintenance underling tumor progression is connected with CSLCs.

Association of wheat chaff derived silica fiber and esophageal cancer in north China

BackgroundDespite decades of research and intervention programs, the epidemic of esophageal squamous cell carcinoma (ESCC) in the Taihang Mountain area of north China has not seen convincing explanation by any risk factor yet and the incidence has not seen a substantial decrease. Based on recently disclosed association of aridity and wheat consumption with esophageal cancer, we revisited the hypothesis of biogenic silica in esophageal cancer development. MethodsFrom the archives of the Pathology Department of Heping Hospital, Changzhi Medical College, we selected three pairs of formalin-fixed samples, tumor tissues and distant normal tissues, of three patients operated for ESCC who had no history of workplace exposure to silica dust. Two pairs of dried tissue samples were used for phytolith (silica body) analysis and another pair for microanalysis with Transmission Electron Microscope (TEM). ResultsOne of the phytoliths in ESCC tumor tissue was similar to the prickle hair on the surface of wheat bract. In the mineral particles detected in the tumor tissue the predominant elements were Si, Ca, and P, whereas Si signals were not obvious in the distant normal tissue. ConclusionsThe preliminary findings on the detection of phytoliths and the higher than normal Si concentration in ESCC tumor tissue warrants further testing the role of biogenic silica in esophageal cancer.

Correlation between methylation of the caveolin‐1 gene and of caveolin‐1 messenger ribonucleic acid, and protein levels and human epidermal growth factor receptor 2 protein expression in adenocarcinomas of the esophagogastric junction

AbstractObjectiveTo investigate the methylation level of caveolin‐1 gene (CAV1) in human adenocarcinomas of the esophagogastric junction (AEG) and distal normal tissues (&gt;10 cm from cancerous foci), and to study the protein expression level of CAV1 and human epidermal growth factor receptor 2 (Her‐2) in AEG and their relationship with CAV1 methylation.MethodsMethylation‐specific polymerase chain reaction was used to detect the methylation level of CAV1 in 67 cases with AEG and distal normal tissues. The mRNA level of CAV1 in AEG and distal normal tissues was detected by reverse transcription polymerase chain reaction. Immunohistochemistry was used to detect the protein expression levels of CAV1 and Her‐2. The correlation between CAV1 methylation, CAV1 mRNA level, CAV1 protein, and Her‐2 protein was further analyzed.ResultsIn AEG, the methylation rate of CAV1 was 28.4% (19/67), and almost no methylation of this gene was found in the distal normal tissues (P &lt; 0.01). The methylation level of CAV1 in carcinoma tissues was related to tumor size, differentiation, and lymph node metastasis in patients (P &lt; 0.05), but was not related to sex, age, the presence of vascular carcinoma thrombus, and clinical stages. The expression rate of CAV1 mRNA and protein in carcinoma tissues was 46.3% and 41.8%, respectively, which was dramatically lower than that in distal normal tissues (P &lt; 0.01). The protein expression level of CAV1 and Her‐2 in carcinoma tissues was correlated with the CAV1 methylation level (P &lt; 0.05).ConclusionsMethylation of CAV1 might be involved in tumor development, and associated with the mRNA and protein expression of CAV1 and Her‐2 protein expression in AEG, which could play an important role in the formation and progression of AEG.

Y-box binding protein-1 promotes tumorigenesis and progression via the epidermal growth factor receptor/AKT pathway in spinal chordoma.

Chordomas are rare bone tumors with a poor prognosis and no approved targeted therapy. Y‐box binding protein‐1 (YBX1) promotes tumor growth, invasion and drug resistance. However, the role of YBX1 in chordoma is unclear. In this study, we examined the expression of YBX1 using immunohistochemistry and found that YBX1 was significantly upregulated in 32 chordoma tissues compared to distant normal tissues. In addition, YBX1 upregulation was associated with surrounding tissue invasion, recurrence and poor prognosis. Biological function studies demonstrated that YBX1 promoted cell proliferation and invasion, accelerated G1/S phase transition, and inhibited apoptosis. Further investigation revealed that YBX1 enhanced epidermal growth factor receptor (EGFR) transcription by directly binding to its promoter in chordoma cells. YBX1 regulated protein expression of p‐EGFR, p‐AKT and its downstream target genes that influenced cell apoptosis, cell cycle transition and cell invasion. YBX1 activated the EGFR/AKT pathway in chordoma and YBX1‐induced elevated expression of key molecules in the EGFR/AKT pathway were downregulated by EGFR and AKT pathway inhibitors. These in vitro results were further confirmed by in vivo data. These data showed that YBX1 promoted tumorigenesis and progression in spinal chordoma via the EGFR/AKT pathway. YBX1 might serve as a prognostic and predictive biomarker, as well as a rational therapeutic target, for chordoma.

Standard CD44 modulates YAP1 through a positive feedback loop in hepatocellular carcinoma

High expression levels of CD44 and YAP have been identified as poor prognostic factors in hepatocellular carcinoma (HCC). However, the mechanistic relationship between CD44 and YAP during HCC tumorigenesis remains largely unknown. To investigate the mutual regulation between standard CD44 (CD44S) and YAP1 in HCC cell lines and tissue samples, CD44S and YAP1 expression in 40 pairs of tumor samples and matched distal normal tissues from HCC patients was examined by immunohistochemical staining. High expression of either CD44S or YAP1 was associated with a younger age and worse pathology grade. In addition, high levels of CD44S and YAP1 were associated with increased vascular invasion and more severe liver cirrhosis, respectively. CD44S expression was positively correlated with YAP1 expression in these HCC tissues. In vitro experiments suggested that CD44S could positively regulate the expression of YAP1 and its target genes via the PI3K/Akt pathway in HCC cells. Moreover, CD44S is regulated by the YAP1/TEAD axis. These results reveal a novel positive feedback loop involving CD44S and YAP1, in which CD44S functions as both an upstream regulator and a downstream effector of YAP1 in HCC. This feedback loop might constitute a broadly conserved module for regulating cell proliferation and invasion during HCC tumorigenesis. Blocking this positive feedback loop that involves CD44S and YAP1 might represent a new approach for HCC treatment.

A Novel Method to Extend a Partial-Body CT for the Reconstruction of Dose to Organs beyond the Scan Range

Epidemiological investigation is an important approach to assessing the risk of late effects after radiotherapy, and organ dosimetry is a crucial part of such analysis. Computed tomography (CT) images, if available, can be a valuable resource for individualizing the dosimetry, because they describe the specific anatomy of the patient. However, CT images acquired for radiation treatment planning purposes cover only a portion of the body near the target volume, whereas for epidemiology, the interest lies in the more distant normal tissues, which may be located outside the scan range. To address this challenge, we developed a novel method, called the Anatomically Predictive Extension (APE), to extend a partial-body CT image stack using images of a computational human phantom matched to the patient based on their height and weight. To test our method, we created five APE phantoms from chest and abdominal images extracted from the chest-abdomen-pelvis (CAP) CT scans of five patients. Organ doses were calculated for simple chest and prostate irradiations that were planned on the reference computational phantom (assumed patient geometry if no CT images are available), APE phantoms (patient-phantom hybrid given a partial-body patient CT) and full patient CAP CT scans (ground truth). The APE phantoms and patient CAP CT scans resulted in nearly identical dosimetry for those organs that were fully included in the partial-body CT used to construct the APE. The calculated doses to these same organs in the reference phantoms differed by up to 20% and 52% for the chest and prostate cases, respectively. For organs outside the scan coverage, the reference phantom showed, on average, dose differences of 31% (chest case) and 41% (prostate case). For the APE phantoms, these values were 26% (chest) and 17% (prostate). The APE method combines patient and phantom images to improve organ dosimetry both inside and outside the scan range. We intend to use the APE method for estimating dose for organs peripheral to the treatment fields; however, this method is quite generalizable with many potential applications.

A Dosimetric Study of a Novel Rotating Gamma Ray System for Stereotactic Body Radiation Therapy

Stereotactic radiosurgery (SRS) unites with current state of art techniques have provided excellent conformity, precision, and versatility for small tumor lesions. Arguably the best known systems are the Gamma ray systems like GammaKnife and the linac based robotic systems like CyberKnife. However, GammaKnife systems are limited to only treating intracranial targets. In this study, we investigate a novel rotating gamma ray system for SRS of intracranial targets and stereotactic body radiation therapy (SBRT) of extracranial body sites as compared to the CyberKnife machine. The RT Pro planning system (version 1.00.4557, Prowess, Concord, CA) was used to generate treatment plans for a prototype CybeRay system for 12 patients, who were previously treated on the CyberKnife. The new CybeRay system (OUR United RT Group., Xian, China) has a rotation gantry containing 16 Co-60 sources focusing at the isocenter. The prototype implemented in RT Pro included 4 different changeable collimators while an improved version included 7 collimators. The treatment can also swing 35° in the superior direction. CybeRay is equipped with an electronic portal imaging device, a cone-beam CT system, and a 6 degrees of freedom couch. Treatment plans from both treatment systems were compared based on isodose distributions, dose volume histograms, conformity index (CI), and the estimated treatment time. CybeRay and CyberKnife results were compared using Wilcoxon signed-rank tests. The planned target volume (PTV) ranged from 2.7cc to 59.5cc. The estimated treatment delivery time for CybeRay plans ranged from 3.7-60.2 minutes as compared to 13-67minutes with Cyberknife (p=0.05). The mean value of CI was 1.23 for Cyberknife as compared to 1.27 for CybeRay (p=0.2). On the other hand, CybeRay was superior with regards to peripheral doses appearing as excessive isodose lines extending to distal normal tissues. To quantify the difference between the two systems, a ring structure (D5cm) was drawn at 5cm distant to the PTV. The maximum dose received by D5cm averaged over 12 patients was 7Gy for CybeRay and 13.4Gy for CyberKnife (p=0.0024). A newly designed rotating gamma ray machine was evaluated through a retrospective dosimetric study using patients previously treated on CyberKnife system. The new machine exhibited high precision stereotaxy and accurate target localization with promising dosimetric capabilities for stereotactic radiation therapy of extra-cranial lesions.Abstract 3565Vol (cc)Dose Gy/fxtreat timeCID5cmCRCKCRCKCRCKAdrenal2736/343.26421.291.188.718.911.850/360.2531.31.236.224.117.745/356.2521.161.2211.415.1Liver4.460/321.2341.191.194.220.44550/528.1381.091.0912.720.3pancreas7.527/311.6441.211.192.85.549.110/131.6381.381.141.53.110.210/145.5331.581.571.33.6Breast25.114/73.69131.211.211112.259.514/74.94191.211.366.28.8Lung12.648/425501.291.188.615.82.754/317.2671.371.259.213.1Mean29401.271.23713.4 Open table in a new tab

High expression of Sam68 in sacral chordomas is associated with worse clinical outcomes.

Src-associated in mitosis of 68 kDa (Sam68), also known as KHDRBS1 (KH domain-containing, RNA-binding, signal transduction-associated 1), is a member of the signal transduction and activation of RNA family. Previous studies have demonstrated that the aberrant expression of Sam68 is associated with the progression and prognosis of a variety of cancers, but little is known about its expression and role in chordomas, which are rare and aggressive bone neoplasms. In this study, we analyzed 40 tumor tissues and 20 distant normal tissues obtained from 40 patients with sacral chordoma using immunohistochemistry, and observed the expression of Sam68 was significantly upregulated in sacral chordomas compared with normal tissues (P=0.001). A positive correlation between the expression of Sam68 and the cell proliferation index Ki-67 was determined using Spearman’s rank correlation test (γ =0.599, P<0.001). In addition, high expression of Sam68 was significantly associated with surrounding muscle invasion (P<0.001). Moreover, Kaplan–Meier curves showed that patients with overexpressed Sam68 had shorter local recurrence-free survival time (P<0.001). Lastly, multivariate analysis indicated that Sam68 is an independent prognostic factor for the local recurrence-free survival of sacral chordomas (hazard ratio =5.929, 95% CI: 1.092–32.188, P=0.039). Our findings suggest the use of Sam68 as a predictor for the recurrence of sacral chordomas.

TBX2 subfamily suppression in lung cancer pathogenesis: a high-potential marker for early detection.

The TBX2 subfamily (TBXs 2, 3, 4 and 5) transactivates or represses genes involved in lung organogenesis. Yet TBX2 subfamily expression in pathogenesis of non-small cell lung cancer (NSCLC), the most common lung malignancy, remains elusive. We sought to probe the expression profile of the TBX2 subfamily in early phases of NSCLC. Expression of TBX2 subfamily was analyzed in datasets of pan-normal specimens as well as NSCLCs and normal lung tissues. TBX2 subfamily expression in matched normal lungs, premalignant hyperplasias and NSCLCs was profiled by transcriptome sequencing. TBX2 subfamily expression was evaluated in the cancerization field consisting of matched NSCLCs and adjacent cytologically-normal airways relative to distant normal lungs and in a dataset of normal bronchial samples from smokers with indeterminate nodules suspicious for malignancy. Statistical analysis was performed using R. TBX2 subfamily expression was markedly elevated in normal lungs relative to other organ-specific normal tissues. Expression of the TBXs was significantly suppressed in NSCLCs relative to normal lungs (P < 10−9). TBX2 subfamily was significantly progressively decreased across premalignant lesions and NSCLCs relative to normal lungs (P < 10−4). The subfamily was significantly suppressed in NSCLCs and adjacent normal-appearing airways relative to distant normal lung tissues (P < 10−15). Further, suppressed TBX2 subfamily expression in normal bronchi was associated with lung cancer status (P < 10−5) in smokers. Our findings suggest that the TBX2 subfamily is notably suppressed in human NSCLC pathogenesis and may serve as a high-potential biomarker for early lung cancer detection in high-risk smokers.

Expression of far upstream element-binding protein 1 correlates with c-Myc expression in sacral chordomas and is associated with tumor progression and poor prognosis

The far upstream element (FUSE)-binding protein 1 (FUBP1), a well-known transcriptional regulator of the proto-oncogene c-Myc, has been demonstrated by previous work to be aberrantly expressed in a variety of tumors and plays a critical role in tumor progression; however, its expression and function in relatively rare and aggressive chordomas remains unclear. In this retrospective study, we reviewed clinicopathologic characteristics of 40 patients diagnosed with sacral chordoma, and analyzed 40 tumor and 20 distant normal tissues obtained from patients during the primary surgical tumor excision. Using immunohistochemistry, we observed an up-regulation in the expression of FUBP1 and c-Myc in sacral chordomas compared with the normal tissues (P = 0.001 for both). Additionally, positive correlations of FUBP1 expression with c-Myc (γ = 0.651, P < 0.001) and the cell proliferation index Ki-67 expression (γ = 0.447, P = 0.004) were indicated using Spearman's rank correlation coefficient. Increased expression of FUBP1 was significantly associated with tumor invasion into the surrounding muscles (P = 0.002). Kaplan-Meier curves demonstrated the association between FUBP1 levels and the patients' local recurrence-free survival (LRFS) (P < 0.001) but not with the overall survival (OS) (P = 0.070). The independent prognostic significance of FUBP1 levels for the LRFS was indicated by multivariate analysis (HR = 4.272; 95% CI, 1.133–16.112; P = 0.032). Our findings demonstrate an association between FUBP1 levels and chordoma progression and prognosis, suggesting that FUBP1 can be used as a biomarker and a potential therapeutic target.

The Expression of Formyl Peptide Receptor 1 is Correlated with Tumor Invasion of Human Colorectal Cancer

Formyl peptide receptors (FPRs) are G protein-coupled chemoattractant receptors expressed mainly in phagocytic leukocytes. High expression of FPRs has also been detected in several cancers but the functions of FPR1 in tumor invasion and metastasis is poorly understood. In this study, we investigated the expression of FPRs in primary human colorectal cancer (CRC) and analyzed the association of FPRs expression with clinicopathological parameters. The levels of FPRs mRNA, especially those of FPR1, were significantly higher in colorectal tumors than in distant normal tissues and adjacent non-tumor tissues. FPR1 mRNA expression was also associated with tumor serosal infiltration. FPR1 protein expression was both in the colorectal epitheliums and tumor infiltrating neutrophils/macrophages. Furthermore, the functions of FPR1 in tumor invasion and tissue repair were investigated using the CRC cell lines SW480 and HT29. Higher cell surface expression of FPR1 is associated with significantly increased migration in SW480 cells compared with HT29 cells that have less FPR1 membrane expression. Finally, genetic deletion of fpr1 increased the survival rate of the resulting knockout mice compared with wild type littermates in a mouse model of colitis-associated colorectal cancer. Our data demonstrate that FPR1 may play an important role in tumor cell invasion in CRC patients.

Elevated S100A8 protein expression in breast cancer cells and breast tumor stroma is prognostic of poor disease outcome.

Elevated S100A8 expression has been observed in cancers of the bladder, esophagus, colon, ovary, and breast. S100A8 is expressed by breast cancer cells as well as by infiltrating immune and myeloid cells. Here we investigate the association of elevated S100A8 protein expression in breast cancer cells and in breast tumor stroma with survival outcomes in a cohort of breast cancer patients. Tissue microarrays (TMA) were constructed from breast cancer specimens from 417 patients with stage I-III breast cancer treated at the University of Michigan Comprehensive Cancer Center between 2004 and 2006. Representative regions of non-necrotic tumor and distant normal tissue from each patient were used to construct the TMA. Automated quantitative immunofluorescence (AQUA) was used to measure S100A8 protein expression, and samples were scored for breast cancer cell and stromal S100A8 expression. S100A8 staining intensity was assessed as a continuous value and by exploratory dichotomous cutoffs. Associations between breast cancer cell and stromal S100A8 expression with disease-free survival and overall survival were determined using the Kaplan-Meier method and Cox proportional hazard models. High breast cancer cell S100A8 protein expression (as indicated by AQUA scores), as a continuous measure, was a significant prognostic factor for OS [univariable hazard ratio (HR) 1.24, 95% confidence interval (CI) 1.00-1.55, p=0.05] in this patient cohort. Exploratory analyses identified optimal S100A8 AQUA score cutoffs within the breast cancer cell and stromal compartments that significantly separated survival curves for the complete cohort. Elevated breast cancer cell and stromal S100A8 expression, indicated by higher S100A8 AQUA scores, significantly associates with poorer breast cancer outcomes, regardless of estrogen receptor status. Elevated breast cancer cell and stromal S1008 protein expression are significant indicators of poorer outcomes in early stage breast cancer patients. Evaluation of S100A8 protein expression may provide additional prognostic information beyond traditional breast cancer prognostic biomarkers.

Abstract 5384: Aberrant methylation of ANK1, the host gene for miR-486, distinguishes lung tumors by histology and smoking status

Abstract MicroRNAs (miRs) regulate many fundamental biological processes primarily through inhibiting the transcription and/or translation of their target genes. MiR-486 is an intragenic miR located within ANK1, a gene that encodes for the adapter protein ankyrin-1. Although the tumor suppressor role of miR-486-5p and its down-regulation in non-small cell lung cancer (NSCLC) is well established, the mechanism(s) leading to its repression is unclear. In this study, we investigated epigenetic regulation of ANK1 and its impact on miR-486-5p expression in lung cancer. DNA methylation and expression levels of ANK1 and miR-486-5p in lung tumor-normal pairs, NSCLC cell lines, as well as various normal blood and lung epithelial cell controls were evaluated using qualitative and quantitative assays. The genome-wide impact of epigenetic repression of ANK1 was evaluated using siRNA. The results show that miR-486-5p expression in lung tumors and NSCLC cell lines was significantly reduced compared to distant normal lung tissue pairs (p = 0.0006) and strongly correlated with ANK1 expression. In NSCLC cell lines with strongly reduced expression of miR-486-5p and ANK1, treatment with the DNA methylation inhibitor 5-aza-2-deoxycytidine significantly increased the expression of both the miR and its host gene. Human ANK1 is a large (~230kb) gene with three promoter regions regulating tissue specific expression of three (ANK1B, ANK1E, and ANK1A) transcript variants. ANK1B (the longest of the three variants) is the primary transcript expressed in lung tissue, while ANK1E and ANK1A are weakly and rarely expressed in lung epithelial cells. ANK1B promoter contains a large CpG island that is methylated in 45% (118/262) of lung tumors but not in normal lung or blood samples. The prevalence for ANK1B methylation was significantly higher (p &amp;lt; 0.001) in lung adenocarcinoma (101/200, 51%) compared to lung squamous cell carcinomas (17/62, 27%) after adjustment for smoking. Comparison of methylation by patients’ smoking history also revealed that ANK1B methylation in lung adenocarcinoma was significantly more prevalent in smokers (57%, 73/128) than in never smokers (39%, 28/72), p = 0.014; HR = 2.086 (CI: 1.157 - 3.759). These findings were confirmed by quantitative methylation assays and independently validated using publicly available methylation data for large (n = 809) NSCLC cases from the Cancer Genome Atlas database. Finally, siRNA mediated knockdown of ANK1 in lung cancer cell lines followed by transcriptome-wide expression and pathway analysis revealed that ANK1 repression primarily alters cancer development and progression pathways. Taken together, our data indicates that aberrant methylation of ANK1B promoter is highly prevalent in lung cancer, discriminates lung tumors by histology and smoking history, and represses the expression of ANK1 along with the tumor-suppressor miR-486-5p it hosts within its last intron. Citation Format: Mathewos Tessema, Christin M. Yingling, Maria A. Picchi, Guodong Wu, Tyron Ryba, Young Lin, Steven A. Belinsky. Aberrant methylation of ANK1, the host gene for miR-486, distinguishes lung tumors by histology and smoking status [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 5384. doi:10.1158/1538-7445.AM2017-5384