Joseph P. McElroy

Comparison of MicroRNA Deep Sequencing of Matched Formalin-Fixed Paraffin-Embedded and Fresh Frozen Cancer Tissues

MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (2–9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson’s r = 0.71–0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified.

A Review of Pulmonary Toxicity of Electronic Cigarettes in the Context of Smoking: A Focus on Inflammation

The use of electronic cigarettes (e-cigs) is increasing rapidly, but their effects on lung toxicity are largely unknown. Smoking is a well-established cause of lung cancer and respiratory disease, in part through inflammation. It is plausible that e-cig use might affect similar inflammatory pathways. E-cigs are used by some smokers as an aid for quitting or smoking reduction, and by never smokers (e.g., adolescents and young adults). The relative effects for impacting disease risk may differ for these groups. Cell culture and experimental animal data indicate that e-cigs have the potential for inducing inflammation, albeit much less than smoking. Human studies show that e-cig use in smokers is associated with substantial reductions in blood or urinary biomarkers of tobacco toxicants when completely switching and somewhat for dual use. However, the extent to which these biomarkers are surrogates for potential lung toxicity remains unclear. The FDA now has regulatory authority over e-cigs and can regulate product and e-liquid design features, such as nicotine content and delivery, voltage, e-liquid formulations, and flavors. All of these factors may impact pulmonary toxicity. This review summarizes current data on pulmonary inflammation related to both smoking and e-cig use, with a focus on human lung biomarkers. Cancer Epidemiol Biomarkers Prev; 26(8); 1175–91. ©2017 AACR.

Molecular-Based Recursive Partitioning Analysis Model for Glioblastoma in the Temozolomide Era: A Correlative Analysis Based on NRG Oncology RTOG 0525

Importance There is a need for a more refined, molecularly based classification model for glioblastoma (GBM) in the temozolomide era. Objective To refine the existing clinically based recursive partitioning analysis (RPA) model by incorporating molecular variables. Design, Setting, and Participants NRG Oncology RTOG 0525 specimens (n = 452) were analyzed for protein biomarkers representing key pathways in GBM by a quantitative molecular microscopy-based approach with semiquantitative immunohistochemical validation. Prognostic significance of each protein was examined by single-marker and multimarker Cox regression analyses. To reclassify the prognostic risk groups, significant protein biomarkers on single-marker analysis were incorporated into an RPA model consisting of the same clinical variables (age, Karnofsky Performance Status, extent of resection, and neurologic function) as the existing RTOG RPA. The new RPA model (NRG-GBM-RPA) was confirmed using traditional immunohistochemistry in an independent data set (n = 176). Main Outcomes and Measures Overall survival (OS). Results In 452 specimens, MGMT (hazard ratio [HR], 1.81; 95% CI, 1.37-2.39; P < .001), survivin (HR, 1.36; 95% CI, 1.04-1.76; P = .02), c-Met (HR, 1.53; 95% CI, 1.06-2.23; P = .02), pmTOR (HR, 0.76; 95% CI, 0.60-0.97; P = .03), and Ki-67 (HR, 1.40; 95% CI, 1.10-1.78; P = .007) protein levels were found to be significant on single-marker multivariate analysis of OS. To refine the existing RPA, significant protein biomarkers together with clinical variables (age, Karnofsky Performance Status, extent of resection, and neurological function) were incorporated into a new model. Of 166 patients used for the new NRG-GBM-RPA model, 97 (58.4%) were male (mean [SD] age, 55.7 [12.0] years). Higher MGMT protein level was significantly associated with decreased MGMT promoter methylation and vice versa (1425.1 for methylated vs 1828.0 for unmethylated; P < .001). Furthermore, MGMT protein expression (HR, 1.84; 95% CI, 1.38-2.43; P < .001) had greater prognostic value for OS compared with MGMT promoter methylation (HR, 1.77; 95% CI, 1.28-2.44; P < .001). The refined NRG-GBM-RPA consisting of MGMT protein, c-Met protein, and age revealed greater separation of OS prognostic classes compared with the existing clinically based RPA model and MGMT promoter methylation in NRG Oncology RTOG 0525. The prognostic significance of the NRG-GBM-RPA was subsequently confirmed in an independent data set (n = 176). Conclusions and Relevance This new NRG-GBM-RPA model improves outcome stratification over both the current RTOG RPA model and MGMT promoter methylation, respectively, for patients with GBM treated with radiation and temozolomide and was biologically validated in an independent data set. The revised RPA has the potential to contribute to improving the accurate assessment of prognostic groups in patients with GBM treated with radiation and temozolomide and to influence clinical decision making. Trial Registration clinicaltrials.gov Identifier: NCT00304031

Patterns of CTCF and ZFHX3 Mutation and Associated Outcomes in Endometrial Cancer

BACKGROUND The genetic events responsible for tumor aggressiveness in endometrioid endometrial cancer (EEC) remain poorly understood. The chromosome 16q22 tumor suppressor genes CTCF and ZFHX3 are both frequently mutated in EEC, but their respective roles in outcome have not been determined. METHODS Targeted deep sequencing of CTCF and ZFHX3 was performed for 542 EEC samples. Copy number loss (CNL) was determined using microsatellite typing of paired tumor and normal DNA and a novel Bayesian method based on variant allele frequencies of germline polymorphisms. All statistical tests were two-sided. RESULTS Mutation rates for CTCF and ZFHX3 were 25.3% and 20.4%, respectively, and there was a statistically significant excess of tumors with mutation in both genes (P = .003). CNL rates were 17.4% for CTCF and 17.2% for ZFHX3, and the majority of CNLs included both CTCF and ZFHX3. Mutations were more frequent in tumors with microsatellite instability, and CNLs were more common in microsatellite-stable tumors (P < .001). Patients with ZFHX3 mutation and/or CNL had higher-grade tumors (P = .001), were older (P < .001), and tended to have more frequent lymphovascular space invasion (P = .07). These patients had reduced recurrence-free and overall survival (RFS: hazard ratio [HR] = 2.35, 95% confidence interval [CI] = 1.38 to 3.99, P = .007; OS: HR = 1.51, 95% CI = 1.11 to 2.07, P = .04). CONCLUSIONS Our data demonstrate there is strong selection for inactivation of both CTCF and ZFHX3 in EEC. Mutation occurs at high frequency in microsatellite-unstable tumors, whereas CNLs are common in microsatellite-stable cancers. Loss of these two tumor suppressors is a frequent event in endometrial tumorigenesis, and ZFHX3 defects are associated with poor outcome.

Epigenetic silencing of MLH1 in endometrial cancers is associated with larger tumor volume, increased rate of lymph node positivity and reduced recurrence-free survival

OBJECTIVES To determine the relationship between mismatch repair (MMR) classification and clinicopathologic features including tumor volume, and explore outcomes by MMR class in a contemporary cohort. METHODS Single institution cohort evaluating MMR classification for endometrial cancers (EC). MMR immunohistochemistry (IHC)±microsatellite instability (MSI) testing and reflex MLH1 methylation testing was performed. Tumors with MMR abnormalities by IHC or MSI and MLH1 methylation were classified as epigenetic MMR deficiency while those without MLH1 methylation were classified as probable MMR mutations. Clinicopathologic characteristics were analyzed. RESULTS 466 endometrial cancers were classified; 75% as MMR proficient, 20% epigenetic MMR defects, and 5% as probable MMR mutations. Epigenetic MMR defects were associated with advanced stage, higher grade, presence of lymphovascular space invasion, and older age. MMR class was significantly associated with tumor volume, an association not previously reported. The epigenetic MMR defect tumors median volume was 10,220mm3 compared to 3321mm3 and 2,846mm3, for MMR proficient and probable MMR mutations respectively (P<0.0001). Higher tumor volume was associated with lymph node involvement. Endometrioid EC cases with epigenetic MMR defects had significantly reduced recurrence-free survival (RFS). Among advanced stage (III/IV) endometrioid EC the epigenetic MMR defect group was more likely to recur compared to the MMR proficient group (47.7% vs 3.4%) despite receiving similar adjuvant therapy. In contrast, there was no difference in the number of early stage recurrences for the different MMR classes. CONCLUSIONS MMR testing that includes MLH1 methylation analysis defines a subset of tumors that have worse prognostic features and reduced RFS.

Discovery and replication of microRNAs for breast cancer risk using genome-wide profiling

Background Genome-wide miRNA expression may be useful for predicting breast cancer risk and/or for the early detection of breast cancer. Results A 41-miRNA model distinguished breast cancer risk in the discovery study (accuracy of 83.3%), which was replicated in the independent study (accuracy = 63.4%, P=0.09). Among the 41 miRNA, 20 miRNAs were detectable in serum, and predicted breast cancer occurrence within 18 months of blood draw (accuracy 53%, P=0.06). These risk-related miRNAs were enriched for HER-2 and estrogen-dependent breast cancer signaling. Materials and Methods MiRNAs were assessed in two cross-sectional studies of women without breast cancer and a nested case-control study of breast cancer. Using breast tissues, a multivariate analysis was used to model women with high and low breast cancer risk (based upon Gail risk model) in a discovery study of women without breast cancer (n=90), and applied to an independent replication study (n=71). The model was then assessed using serum samples from the nested case-control study (n=410). Conclusions Studying breast tissues of women without breast cancer revealed miRNAs correlated with breast cancer risk, which were then found to be altered in the serum of women who later developed breast cancer. These results serve as proof-of-principle that miRNAs in women without breast cancer may be useful for predicting breast cancer risk and/or as an adjunct for breast cancer early detection. The miRNAs identified herein may be involved in breast carcinogenic pathways because they were first identified in the breast tissues of healthy women.

Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin–positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a KrasG12D-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2–deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic KrasG12D signaling during the initial stages of tumor development.

Analysis of the exome aggregation consortium (ExAC) database suggests that the BAP1-tumor predisposition syndrome is underreported in cancer patients

The BAP1‐tumor predisposition syndrome (BAP1‐TPDS) has been recently identified to predispose patients to a variety of cancers and preneoplastic lesions. About 130 unrelated probands have been identified worldwide; however, the impact of the syndrome is suspected to be much larger given the diversity of the cancer phenotype. To evaluate the frequency of germline BAP1 mutations in the general and cancer populations, we analyzed the Exome Aggregation Consortium (ExAC), a database that contains 53105 exomes of unrelated individuals unaffected by cancer (general population) and exomes of 7601 unrelated individuals affected by cancer provided by the Cancer Genome Atlas (TCGA, cancer subjects). BAP1 null variants were seen at much higher frequency in the cancer subjects (0.0526%) compared to the general population (0.00188%) with a relative risk of 27.93 and (P = 0.0011, [95% CI: 3.122‐249.883], Fishers exact test). We also studied a reported BAP1 null variant, c.1203T > G, p.T401* (rs200156887), observed commonly in the general population. Sequencing and restriction fragment polymorphism of the RT‐4 cell line that contains this variant revealed that it is in fact a 3bp deletion/insertion, c.1201_1203delinsGAG, a likely benign missense alteration p.Y401E explaining the relative high frequency of this variant in the general population. In conclusion, germline null mutations in BAP1 have a significantly higher frequency in cancer patients than the general population. Given the low frequency of reported families with BAP1‐TPDS, our results suggest that the syndrome is underreported especially in patients with cancer.

Landscape of genome-wide age-related DNA methylation in breast tissue

Despite known age-related DNA methylation (aDNAm) changes in breast tumors, little is known about aDNAm in normal breast tissues. Breast tissues from a cross-sectional study of 121 cancer-free women, were assayed for genome-wide DNA methylation. mRNA expression was assayed by microarray technology. Analysis of covariance was used to identify aDNAm’s. Altered methylation was correlated with expression of the corresponding gene and with DNA methyltransferase protein DNMT3A, assayed by immunohistochemistry. Publically-available TCGA-BRCA data were used for replication. 1,214 aDNAm’s were identified; 97% with increased methylation, and all on autosomes. Sites with increased methylation were predominantly in CpG lslands and non-enhancers. aDNAm’s with decreased methylation were generally located in intergenic regions, non-CpG Islands, and enhancers. Of the aDNAm’s identified, 650 are known to be involved in cancer, including ESR1 and beta-estradiol responsive genes. Expression of DNMT3A was positively associated with age. Two aDNAm’s showed borderline significant associations with DNMT3A expression; KRR1 (OR 6.57, 95% CI: 2.51–17.23) and DHRS12 (OR 6.08, 95% CI: 2.33–15.86). A subset of aDNAm’s co-localized within vulnerable regions for somatic mutations in cancers including breast cancer. Expression of C19orf48 was inversely and significantly correlated with its methylation level. In the TCGA dataset, 84% and 64% of the previously identified aDNAm’s were correlated with age in both normal-adjacent and tumor breast tissues, with differential associations by histological subtype. Given the similarity of findings in the breast tissues of healthy women and breast tumors, aDNAm’s may be one pathway for increased breast cancer risk with age.

Genetic variants in thyroid cancer distant metastases

Thyroid carcinoma is the most rapidly increasing solid tumor in the United States; although there has been a great emphasis on the analysis of primary tumors, predictors of both late-stage progression and response to therapy are more poorly defined due in part to the scarcity of progressive metastatic tissues. Therapeutic evidence of mixed responses in metastatic lesions and the limited available genomic data suggest that distant metastases in thyroid cancer are heterogeneous, driven by known oncogenes and other pathways that also might be therapeutic targets. We analyzed the genomes of a small number of rare surgically resected metastatic thyroid cancer lesions along with paired normal and primary tumor samples when available, in an effort to better characterize progressive distant metastases. The findings confirm the presence of mutations to known tumor drivers (BRAF and RAS) in metastatic samples. The results also identified the co-occurrence in predicted functional variants to the DNA damage repair (DDR) genes ATM and ERCC4 in metastatic lesions that did not show alterations of the MAPK pathway. We examined the exomes of 19 samples (including 5 paired normal tissues) from 11 follicular cellderived thyroid cancer patients with surgically resected distant metastases by custom exomeSeq (Supplementary Materials and Methods, see section on supplementary data given at the end of this article). Tumors with different histopathologies were included and were confirmed by an expert thyroid pathologist (PW). All patients were treated with TSH suppression, eight of them received I-131 therapy and none of them received chemotherapy or kinase inhibitors. Overall, 19,299 unique variants in the 682 genes and genomic regions were identified by an exomeSeq custom panel. We sought to identify rare, conserved, exonic variants that were likely to have functional effects. Thus, we focused on 1742 exonic variants in exons of sequenced genes. We excluded synonymous variants and variants with higher than 0.01 minor allelic frequency in the 1000 Genomes Project. We then filtered to include variants located in regions that were conserved through vertebrate evolution. These criteria produced a list of 349 variants in 199 genes. Unsupervised clustering using complete linkage and Euclidian distance identified clusters based on patients, suggesting that most variants were primarily associated with individuals rather than histology or tissue location (data not shown). We finally applied a filter to identify variants predicted or known to have a damaging (SIFT) or deleterious (PolyPhen) effect on the protein. Figure 1 summarizes the filtering strategy. In the primary tumors of patients with distant metastases, 33 variants in 31 genes were identified by comparing with normal samples. We focused on genes affected in more than one tumor sample and found three such genes: BCR, BRAF and MAP4 (Fig. 2). Two variants that affect BRAF in five primary tumor samples were identified. A Hürthle cell carcinoma (HCC) bore a BRAF T241M variant, although its functional significance is uncertain. The remaining samples with BRAF had mutations resulting in BRAF V600E and included two papillary thyroid cancers (PTC) primary tumors and a metastatic lesion, and one anaplastic thyroid cancer (ATC) metastatic lesion. We also identified four samples from four patients with an identical insertion resulting in a frameshift (chr22:23653975 Indel: TCCGG) in BCR including three primary tumors and one metastasis. The primary tumor samples were from follicular (FTC) and HCC, and a metastatic lesion from BRAF V600E PTC. Finally, three primary tumors had an identical mutation (R1112P) in MAP4. Two of the tumors are from patients with HCC, and the third is a PTC with a concurrent BRAFV600E mutation. BCR and MAP4 are functionally involved in cytoskeletal dynamics, although those roles and functional implications of the variants identified are not completely characterized. In the metastatic tissues, we identified thirty variants in twenty-eight genes after filtering unique vs normal tissues. Variants in three genes, ATM, BRAF and ERCC4,