Publications by authors named "Leslie Cope"

124 Publications

DNA-methylation for the detection and distinction of 19 human malignancies.

Epigenetics 2021 Mar 5:1-11. Epub 2021 Mar 5.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

The contribution of DNA-methylation based gene silencing to carcinogenesis is well established. Increasingly, DNA-methylation is examined using genome-wide techniques, with recent public efforts yielding immense data sets of diverse malignancies representing the vast majority of human cancer related disease burden. Whereas mutation events may group preferentially or in high frequency with a given histology, mutations are poor classifiers of tumour type. Here we examine the hypothesis that cancer-specific DNA-methylation reflects the tissue of origin or carcinogenic risk factor, and these methylation abnormalities may be used to faithfully classify tumours according to histology. We present an analysis of 7427 tumours representing 19 human malignancies and 708 normal samples demonstrating that specific tumour changes in methylation can correctly determine site of origin and tumour histology with 86% overall accuracy. Examination of misclassified tumours reveals underlying shared biology as the source of misclassifications, including common cell of origin or risk factors.
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http://dx.doi.org/10.1080/15592294.2021.1890885DOI Listing
March 2021

HEYL Regulates Neoangiogenesis Through Overexpression in Both Breast Tumor Epithelium and Endothelium.

Front Oncol 2020 15;10:581459. Epub 2021 Jan 15.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Blocking tumor angiogenesis is an appealing therapeutic strategy, but to date, success has been elusive. We previously identified HEYL, a downstream target of Notch signaling, as an overexpressed gene in both breast cancer cells and as a tumor endothelial marker, suggesting that HEYL overexpression in both compartments may contribute to neoangiogenesis. Carcinomas arising in double transgenic Her2-neu/HeyL mice showed higher tumor vessel density and significantly faster growth than tumors in parental Her2/neu mice. Providing mechanistic insight, microarray-based mRNA profiling of HS578T-tet-off-HEYL human breast cancer cells revealed upregulation of several angiogenic factors including CXCL1/2/3 upon HEYL expression, which was validated by RT-qPCR and protein array analysis. Upregulation of the cytokines CXCL1/2/3 occurred through direct binding of HEYL to their promoter sequences. We found that vessel growth and migration of human vascular endothelial cells (HUVECs) was promoted by conditioned medium from HS578T-tet-off-HEYL carcinoma cells, but was blocked by neutralizing antibodies against CXCL1/2/3. Supporting these findings, suppressing HEYL expression using shRNA in MDA-MB-231 cells significantly reduced tumor growth. In addition, suppressing the action of proangiogenic cytokines induced by HEYL using a small molecule inhibitor of the CXCl1/2/3 receptor, CXCR2, in combination with the anti-VEGF monoclonal antibody, bevacizumab, significantly reduced tumor growth of MDA-MB-231 xenografts. Thus, HEYL expression in tumor epithelium has a profound effect on the vascular microenvironment in promoting neoangiogenesis. Furthermore, we show that lack of HEYL expression in endothelial cells leads to defects in neoangiogenesis, both under normal physiological conditions and in cancer. Thus, HeyL-/- mice showed impaired vessel outgrowth in the neonatal retina, while the growth of mammary tumor cells E0771 was retarded in syngeneic HeyL-/- mice compared to wild type C57/Bl6 mice. Blocking HEYL's angiogenesis-promoting function in both tumor cells and tumor-associated endothelium may enhance efficacy of therapy targeting the tumor vasculature in breast cancer.
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http://dx.doi.org/10.3389/fonc.2020.581459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845423PMC
January 2021

Supervised mutational signatures for obesity and other tissue-specific etiological factors in cancer.

Elife 2021 Jan 25;10. Epub 2021 Jan 25.

Division of Biostatistics and Bioinformatics, Department of Oncology, Sidney Kimmel Cancer Center, Johns Hopkins University School of Medicine, Baltimore, United States.

Determining the etiologic basis of the mutations that are responsible for cancer is one of the fundamental challenges in modern cancer research. Different mutational processes induce different types of DNA mutations, providing 'mutational signatures' that have led to key insights into cancer etiology. The most widely used signatures for assessing genomic data are based on unsupervised patterns that are then retrospectively correlated with certain features of cancer. We show here that supervised machine-learning techniques can identify signatures, called SuperSigs, that are more predictive than those currently available. Surprisingly, we found that aging yields different SuperSigs in different tissues, and the same is true for environmental exposures. We were able to discover SuperSigs associated with obesity, the most important lifestyle factor contributing to cancer in Western populations.
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http://dx.doi.org/10.7554/eLife.61082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872524PMC
January 2021

Integrated Multiparametric Radiomics and Informatics System for Characterizing Breast Tumor Characteristics with the OncotypeDX Gene Assay.

Cancers (Basel) 2020 Sep 27;12(10). Epub 2020 Sep 27.

Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

Optimal use of multiparametric magnetic resonance imaging (mpMRI) can identify key MRI parameters and provide unique tissue signatures defining phenotypes of breast cancer. We have developed and implemented a new machine-learning informatic system, termed Informatics Radiomics Integration System (IRIS) that integrates clinical variables, derived from imaging and electronic medical health records (EHR) with multiparametric radiomics (mpRad) for identifying potential risk of local or systemic recurrence in breast cancer patients. We tested the model in patients ( = 80) who had Estrogen Receptor positive disease and underwent OncotypeDX gene testing, radiomic analysis, and breast mpMRI. The IRIS method was trained using the mpMRI, clinical, pathologic, and radiomic descriptors for prediction of the OncotypeDX risk score. The trained mpRad IRIS model had a 95% and specificity was 83% with an Area Under the Curve (AUC) of 0.89 for classifying low risk patients from the intermediate and high-risk groups. The lesion size was larger for the high-risk group (2.9 ± 1.7 mm) and lower for both low risk (1.9 ± 1.3 mm) and intermediate risk (1.7 ± 1.4 mm) groups. The lesion apparent diffusion coefficient (ADC) map values for high- and intermediate-risk groups were significantly ( < 0.05) lower than the low-risk group (1.14 vs. 1.49 × 10 mm/s). These initial studies provide deeper insight into the clinical, pathological, quantitative imaging, and radiomic features, and provide the foundation to relate these features to the assessment of treatment response for improved personalized medicine.
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http://dx.doi.org/10.3390/cancers12102772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601838PMC
September 2020

Integrative Transcriptome Analyses of the Human Fallopian Tube: Fimbria and Ampulla-Site of Origin of Serous Carcinoma of the Ovary.

Cancers (Basel) 2020 Apr 27;12(5). Epub 2020 Apr 27.

Division of Gynecologic Oncology, Sylvester Comprehensive Cancer Center, Leonard Miller School of Medicine, University of Miami, 1550 NW 10th Ave, Miami, 33134 FL, USA.

Epithelial ovarian cancer represents a group of heterogeneous diseases with high grade serous cancer (HGSC) representing the most common histotype. Molecular profiles of precancerous lesions found in the fallopian tube have implicated this tissue as the presumptive site of origin of HGSC. Precancerous lesions are primarily found in the distal fallopian tube (fimbria), near the ovary relative to the proximal tissue (ampulla), nearer to the uterus. The proximity of the fimbria to the ovary and the link between ovulation, through follicular fluid release, and ovarian cancer risk led us to examine transcriptional responses of fallopian tube epithelia (FTE) at the different anatomical sites of the human fallopian tube. Gene expression profiles of matched FTE from the fimbria and from premenopausal women resulted in differentially expressed genes (DEGs): CYYR1, SALL1, FOXP2, TAAR1, AKR1C2/C3/C4, NMBR, ME1 and GSTA2. These genes are part of the antioxidant, stem and inflammation pathways. Comparisons between the luteal phase (post-ovulation) to the follicular phase (pre-ovulation) demonstrated greater differences in DEGs than a comparison between fimbria and fallopian tube anatomical differences alone. This data suggests that cyclical transcriptional changes experienced in pre-menopause are inherent physiological triggers that expose the FTE in the fimbria to cytotoxic stressors. These cyclical exposures induce transcriptional changes reflective of genotoxic and cytotoxic damage to the FTE in the fimbria which are closely related to transcriptional and genomic alterations observed in ovarian cancer.
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http://dx.doi.org/10.3390/cancers12051090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281286PMC
April 2020

DNA methylation markers predict recurrence-free interval in triple-negative breast cancer.

NPJ Breast Cancer 2020 31;6. Epub 2020 Jan 31.

1Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.

We lack tools to risk-stratify triple-negative breast cancer (TNBC). Our goal was to develop molecular tools to predict disease recurrence. Methylation array analysis was performed on 110 samples treated by locoregional therapy obtained from institutional cohorts. Discovered marker sets were then tested by Kaplan-Meier analyses in a prospectively collected TNBC cohort of 49 samples from the no-chemotherapy arms of IBCSG trials VIII and IX, and by logistic regression in a chemotherapy-treated cohort of 121 TNBCs from combined IBCSG trials and institutional repositories. High methylation was associated with shorter recurrence-free interval in the no-chemotherapy arm of the IBCSG studies, as well as in the chemotherapy-treated patients within the combined institutional and IBCSG chemotherapy cohorts (100 marker panel,  = 0.002; 30 marker panel,  = 0.05). Chromosome 19 sites were enriched among these loci. In conclusion, our hypermethylation signatures identify increased recurrence risk independent of whether patients receive chemotherapy.
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http://dx.doi.org/10.1038/s41523-020-0145-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994477PMC
January 2020

Functional Loss of and Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells.

Mol Cancer Res 2019 12 14;17(12):2480-2491. Epub 2019 Oct 14.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRX cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRX cells by introducing mutations in the locus, the essential RNA component of telomerase. These LAPC-4 ATRX TERC cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. IMPLICATIONS: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891209PMC
December 2019

High mobility group A1 (HMGA1) protein and gene expression correlate with ER-negativity and poor outcomes in breast cancer.

Breast Cancer Res Treat 2020 Jan 17;179(1):25-35. Epub 2019 Sep 17.

Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

Purpose: The high mobility group A1 (HMGA1) chromatin remodeling protein is required for metastatic progression and cancer stem cell properties in preclinical breast cancer models, although its role in breast carcinogenesis has remained unclear. To investigate HMGA1 in primary breast cancer, we evaluated immunoreactivity score (IRS) in tumors from a large cohort of Asian women; HMGA1 gene expression was queried from two independent Western cohorts.

Methods: HMGA1 IRS was generated from breast tumors in Korean women as the product of staining intensity (weak = 1, moderate = 2, strong = 3) and percent positive cells (< 5% = 0, 5-30% = 1, 30-60% = 2, > 60% = 3), and stratified into three groups: low (< 3), intermediate (3-6), high (> 6). We assessed HMGA1 and estrogen receptor (ESR1) gene expression from two large databases (TCGA, METABRIC). Overall survival was ascertained from the METABRIC cohort.

Results: Among 540 primary tumors from Korean women (181 ER-negative, 359 ER-positive), HMGA1 IRS was < 3 in 89 (16.5%), 3-6 in 215 (39.8%), and > 6 in 236 (43.7%). High HMGA1 IRS was associated with estrogen receptor (ER)-negativity (χ = 12.07; P = 0.002) and advanced nuclear grade (χ = 12.83; P = 0.012). In two large Western cohorts, the HMGA1 gene was overexpressed in breast cancers compared to non-malignant breast tissue (P < 0.0001), including Asian, African American, and Caucasian subgroups. HMGA1 was highest in ER-negative tumors and there was a strong inverse correlation between HMGA1 and ESR1 gene expression (Pearson r = - 0.60, P < 0.0001). Most importantly, high HMGA1 predicted decreased overall survival (P < 0.0001) for all women with breast cancer and further stratified ER-positive tumors into those with inferior outcomes.

Conclusions: Together, our results suggest that HMGA1 contributes to estrogen-independence, tumor progression, and poor outcomes. Moreover, further studies are warranted to determine whether HMGA1 could serve as a prognostic marker and therapeutic target for women with breast cancer.
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http://dx.doi.org/10.1007/s10549-019-05419-1DOI Listing
January 2020

Genome-wide investigation of intragenic DNA methylation identifies ZMIZ1 gene as a prognostic marker in glioblastoma and multiple cancer types.

Int J Cancer 2019 12 9;145(12):3425-3435. Epub 2019 Aug 9.

Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD.

DNA methylation has long been recognized as a tumor-promoting factor when aberrantly regulated in the promoter region of genes. However, the effect of intragenic DNA methylation remains poorly understood on the clinical aspects of cancer. Here, we first evaluated the significance of intragenic DNA methylation for survival outcomes of cancer patients in a genome-wide manner. Glioblastoma patients with hypermethylated intragenic regions exhibited better survival than hypomethylated patients. Enrichment analyses of intragenic DNA methylation profiles with epigenetic signatures prioritized the intragenic DNA methylation of ZMIZ1 as a possible glioblastoma prognostic marker that is independent of MGMT methylation in IDH1 wild-type patients. This intragenic region harbored molecular signatures of alternative transcription across many cell types. Furthermore, we found that the intragenic region of ZMIZ1 can serve as a molecular marker in multiple cancers including astrocytomas, bladder cancer and renal cell carcinoma according to DNA methylation status. Finally, in vitro and in vivo experiments uncovered the role of ZMIZ1 as a driver of tumor cell migration. Altogether, our results identify ZMIZ1 as a prognostic marker in cancer and highlight the clinical significance of intragenic methylation in cancer.
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http://dx.doi.org/10.1002/ijc.32587DOI Listing
December 2019

Clinicopathologic and Molecular Features of Paired Cases of Metachronous Ovarian Serous Borderline Tumor and Subsequent Serous Carcinoma.

Am J Surg Pathol 2019 11;43(11):1462-1472

Departments of Pathology.

Although risk factors have been established for the development of serous carcinoma after a diagnosis of serous borderline tumor (SBT), comprising atypical proliferative serous tumor (APST) (ie, conventional SBT) and noninvasive low-grade serous carcinoma (niLGSC) (ie, micropapillary SBT), subsequent invasive carcinoma still occurs in a subset of women who are not at increased risk. Whether subsequent serous carcinoma in women with a prior SBT represents malignant progression/recurrence or an independent primary tumor is unclear, and the combined clinicopathologic and molecular features of SBTs and their subsequent carcinomas have not been fully characterized. In this study, we analyzed a cohort of 42 women initially diagnosed with SBT who subsequently developed serous carcinoma of a total of 1025 cases of ovarian SBT from a nationwide population-based cohort. Review of the diagnostic slides was performed from this subset of SBTs and matched metachronous invasive serous carcinomas (39 low grade, 3 high grade). DNA was extracted from tissue blocks available for 41 cases (both SBT and carcinoma, n=36; SBT only, n=3; carcinoma only, n=2). Samples were subjected to digital droplet PCR to analyze mutation hotspots in KRAS (codon 12) and BRAF (V600E), which are frequently found in low-grade serous tumors. Eighty-one percent of SBTs (34/42) were APST, and 19% (8/42) were niLGSC. Forty percent of cases (17/42) were FIGO stage I, the majority of which were APST (14/17; 82%). The median time to development of carcinoma was 9 years (range, 0.6 to 25 y). Mutations in SBTs were distributed as follows: 5/39 (13%) BRAF mutant, 22/39 (56%) KRAS mutant, and 12/39 (31%) wild-type for both genes. There was a significant relationship between SBT gene mutation and histologic type, with BRAF mutations occurring exclusively in APST and a higher frequency of niLGSC among SBTs wild-type for BRAF and KRAS (P=0.01). The diffuse presence of tumor cells with abundant eosinophilic cytoplasm was significantly associated with the BRAF mutation (P=0.001). Mutational analyses of matched SBT/carcinoma pairs revealed concordant profiles in 33/36 (92%) cases, of which 19 (53%) were KRAS mutant, 4 (11%) were BRAF mutant, and 10 (28%) were wild type for both genes. The 3 discordant cases consisted of a wild-type niLGSC with a subsequent BRAF-mutant invasive LGSC, a KRAS-mutant APST with a KRAS-mutant LGSC, and a BRAF-mutant APST with subsequent development of a KRAS-mutant high-grade serous carcinoma. In conclusion, some women with SBTs can subsequently develop serous carcinoma, occasionally over 10 years later. Most subsequent carcinomas are low grade, but a small subset can be high grade. The type of gene mutation in SBT correlates with various histologic features. While most cases of serous carcinoma developing after a diagnosis of SBT probably represent tumor progression, a minority are independent primary tumors, presumably arising from endosalpingiosis.
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http://dx.doi.org/10.1097/PAS.0000000000001325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786947PMC
November 2019

DNA Methylation Markers for Breast Cancer Detection in the Developing World.

Clin Cancer Res 2019 11 12;25(21):6357-6367. Epub 2019 Jul 12.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: An unmet need in low-resource countries is an automated breast cancer detection assay to prioritize women who should undergo core breast biopsy and pathologic review. Therefore, we sought to identify and validate a panel of methylated DNA markers to discriminate between cancer and benign breast lesions using cells obtained by fine-needle aspiration (FNA). Two case-control studies were conducted comparing cancer and benign breast tissue identified from clinical repositories in the United States, China, and South Africa for marker selection/training ( = 226) and testing ( = 246). Twenty-five methylated markers were assayed by Quantitative Multiplex-Methylation-Specific PCR (QM-MSP) to select and test a cancer-specific panel. Next, a pilot study was conducted on archival FNAs (49 benign, 24 invasive) from women with mammographically suspicious lesions using a newly developed, 5-hour, quantitative, automated cartridge system. We calculated sensitivity, specificity, and area under the receiver-operating characteristic curve (AUC) compared with histopathology for the marker panel.

Results: In the discovery cohort, 10 of 25 markers were selected that were highly methylated in breast cancer compared with benign tissues by QM-MSP. In the independent test cohort, this panel yielded an AUC of 0.937 (95% CI = 0.900-0.970). In the FNA pilot, we achieved an AUC of 0.960 (95% CI = 0.883-1.0) using the automated cartridge system.

Conclusions: We developed and piloted a fast and accurate methylation marker-based automated cartridge system to detect breast cancer in FNA samples. This quick ancillary test has the potential to prioritize cancer over benign tissues for expedited pathologic evaluation in poorly resourced countries.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825533PMC
November 2019

DNA methylation genome-wide analysis in remnant and primary gastric cancers.

Gastric Cancer 2019 11 12;22(6):1109-1120. Epub 2019 Mar 12.

Department of Surgery, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 600N. Wolfe Street, Blalock 240, Baltimore, MD, 21287, USA.

Background: Although primary (PGC) and remnant gastric cancers (RGC) both originate from the same gastrointestinal organ, they have very distinct clinicopathological behaviors. We hypothesized that there would be distinct differences in DNA methylation patterns that would occur during carcinogenesis of RGC and PGC, and that the differences in methylation patterns may help identify the primary factor contributing to chronic inflammation in patients with RGC.

Methods: We investigated the genome-wide DNA methylation patterns of PGC and RGC tissues from 48 patients using the Infinium HumanMethylation450 Beadchip assay. The results were validated by quantitative methylation-specific PCR (qMSP) in separate, independent cohorts.

Results: We found that in our training cohort of 48 patients, the most variable genes from the gastric cancer tissues identified by the Infinium HumanMethylation450 Beadchip clustered the resultant heatmap into high and low methylation groups. On multivariate analysis, PGCs contributed significantly to the high methylation group (p = 0.004, OR 12.33), which suggested that the promoter methylation status in PGC is higher than that in RGC. Supporting this conclusion was the finding that in a separate qMSP analysis in a test cohort, the EPB41L3 gene, chosen because of its high β value on microarray analysis in the gastric cancer tissues, had significantly higher DNA promoter methylation in cancer tissues in the validation PGC tissues than in RGC.

Conclusions: This study demonstrated that promoter methylation status in PGC is higher than in RGC. This result may reflect the effects of the absence of Helicobacter pylori on the reduced DNA methylation in the remnant stomach.
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http://dx.doi.org/10.1007/s10120-019-00949-5DOI Listing
November 2019

Measuring DNA Copy Number Variation Using High-Density Methylation Microarrays.

J Comput Biol 2019 04 21;26(4):295-304. Epub 2019 Feb 21.

5 Department of Oncology Bioinformatics, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

Genetic and epigenetic changes drive carcinogenesis, and their integrated analysis provides insights into mechanisms of cancer development. Computational methods have been developed to measure copy number variation (CNV) from methylation array data, including ChAMP-CNV, CN450K, and, introduced here, Epicopy. Using paired single nucleotide polymorphism (SNP) and methylation array data from the public The Cancer Genome Atlas repository, we optimized CNV calling and benchmarked the performance of these methods. We optimized the thresholds of all three methods and showed comparable performance across methods. Using Epicopy as a representative analysis of Illumina450K array, we show that Illumina450K-derived CNV methods achieve a sensitivity of 0.7 and a positive predictive value of 0.75 in identifying CNVs, which is similar to results achieved when comparing competing SNP microarray platforms with each other.
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http://dx.doi.org/10.1089/cmb.2018.0143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479247PMC
April 2019

Persistent mutant oncogene specific T cells in two patients benefitting from anti-PD-1.

J Immunother Cancer 2019 02 11;7(1):40. Epub 2019 Feb 11.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD, USA.

Background: Several predictive biomarkers are currently approved or are under investigation for the selection of patients for checkpoint blockade. Tumor PD-L1 expression is used for stratification of non-small cell lung (NSCLC) patients, with tumor mutational burden (TMB) also being explored with promising results, and mismatch-repair deficiency is approved for tumor site-agnostic disease. While tumors with high PD-L1 expression, high TMB, or mismatch repair deficiency respond well to checkpoint blockade, tumors with lower PD-L1 expression, lower mutational burdens, or mismatch repair proficiency respond much less frequently.

Case Presentation: We studied two patients with unexpected responses to checkpoint blockade monotherapy: a patient with PD-L1-negative and low mutational burden NSCLC and one with mismatch repair proficient colorectal cancer (CRC), both of whom lack the biomarkers associated with response to checkpoint blockade, yet achieved durable clinical benefit. Both maintained T-cell responses in peripheral blood to oncogenic driver mutations - BRAF-N581I in the NSCLC and AKT1-E17K in the CRC - years after treatment initiation. Mutation-specific T cells were also found in the primary tumor and underwent dynamic perturbations in the periphery upon treatment.

Conclusions: These findings suggest that T cell responses to oncogenic driver mutations may be more prevalent than previously appreciated and could be harnessed in immunotherapeutic treatment, particularly for patients who lack the traditional biomarkers associated with response. Comprehensive studies are warranted to further delineate additional predictive biomarkers and populations of patients who may benefit from checkpoint blockade.
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http://dx.doi.org/10.1186/s40425-018-0492-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371497PMC
February 2019

Induction of cell cycle arrest and inflammatory genes by combined treatment with epigenetic, differentiating, and chemotherapeutic agents in triple-negative breast cancer.

Breast Cancer Res 2018 11 28;20(1):145. Epub 2018 Nov 28.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: A combination of entinostat, all-trans retinoic acid, and doxorubicin (EAD) induces cell death and differentiation and causes significant regression of xenografts of triple-negative breast cancer (TNBC).

Methods: We investigated the mechanisms underlying the antitumor effects of each component of the EAD combination therapy by high-throughput gene expression profiling of drug-treated cells.

Results: Microarray analysis showed that entinostat and doxorubicin (ED) altered expression of genes related to growth arrest, inflammation, and differentiation. ED downregulated MYC, E2F, and G2M cell cycle genes. Accordingly, entinostat sensitized the cells to doxorubicin-induced growth arrest at G2. ED induced interferon genes, which correlated with breast tumors containing a higher proportion of tumor-infiltrating lymphocytes. ED also increased the expression of immune checkpoint agonists and cancer testis antigens. Analysis of TNBC xenografts showed that EAD enhanced the inflammation score in nude mice. Among the genes differentially regulated between the EAD and ED groups, an all-trans retinoic acid (ATRA)-regulated gene, DHRS3, was induced in EAD-treated xenografts. DHRS3 was expressed at lower levels in human TNBC metastases compared to normal breast or primary tumors. High expression of ED-induced growth arrest and inflammatory genes was associated with better prognosis in TNBC patients.

Conclusions: Entinostat potentiated doxorubicin-mediated cell death and the combination induced inflammatory signatures. The ED-induced immunomodulation may improve immunotherapy. Addition of ATRA to ED may potentiate inflammation and contribute to TNBC regression.
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http://dx.doi.org/10.1186/s13058-018-1068-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6263070PMC
November 2018

Young age at diagnosis is associated with worse prognosis in the Luminal A breast cancer subtype: a retrospective institutional cohort study.

Breast Cancer Res Treat 2018 Dec 17;172(3):689-702. Epub 2018 Sep 17.

Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.

Purpose: Although age is a recognized independent prognostic risk factor, its relative importance among molecular subtypes of Breast cancer (BCA) is not well documented. The aim of this study was to evaluate the prognostic role of age at diagnosis among different immunohistochemical subtypes of BCA.

Methods: We conducted a retrospective study of women with invasive BCA undergoing surgery at the Johns Hopkins Hospital, excluding patients presenting with stage IV breast cancer. Patients were stratified into three age groups: ≤ 40, 41-60, and > 60 years, and multivariable analysis was performed using Cox regression. We also identified differentially expressed genes (DEG) between age groups among BCA subtypes in the public TCGA dataset. Finally, we identified key driver genes within the DEGs using a weighted gene co-expression network analysis.

Results: Luminal A breast cancer patients had significantly lower 5 year disease-free survival (DFS) and distant metastasis-free survival (DMFS) in the ≤ 40 year age group compared to the 41-60 year age group, while the other molecular subtypes showed no significant association of DFS or DMFS with age. Age was a stronger outcome predictor than tumor grade or proliferative index in Luminal A BCA patients, but not other subtypes. BCA TCGA gene expression data were divided into two groups (≤ 40 years, > 40 years). We identified 374 DEGs in the Luminal A BCA subset, which were enriched in seven pathways and two modules of co-expressed genes. No age group-specific DEGs were identified in non-Luminal A subtypes.

Conclusions: Age at diagnosis may be an important prognostic factor in Luminal A BCA.
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http://dx.doi.org/10.1007/s10549-018-4950-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786966PMC
December 2018

Methylomic Analysis of Ovarian Cancers Identifies Tumor-Specific Alterations Readily Detectable in Early Precursor Lesions.

Clin Cancer Res 2018 12 14;24(24):6536-6547. Epub 2018 Aug 14.

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages.

Experimental Design: MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women.

Results: Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value ≥ 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes ( and ) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes.

Conclusions: A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295225PMC
December 2018

The Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST) Assay: A Sensitive Platform for Monitoring Antitumor Immunity.

Cancer Immunol Res 2018 08 12;6(8):888-899. Epub 2018 Jun 12.

The Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Mutation-associated neoantigens (MANA) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing cytokine production such as ELISpot and intracellular cytokine staining have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates T-cell receptor sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigen-specific clonotypic amplifications. This assay can be adapted for all types of antigens, including MANAs via tumor exome-guided prediction of MANAs. Following identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices. .
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072595PMC
August 2018

A Standardized Needs Assessment Tool to Inform the Curriculum Development Process for Pediatric Resuscitation Simulation-Based Education in Resource-Limited Settings.

Front Pediatr 2018 28;6:37. Epub 2018 Feb 28.

Department of Emergency Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Introduction: Under five mortality rates (UFMR) remain high for children in low- and middle-income countries (LMICs) in the developing world. Education for practitioners in these environments is a key factor to improve outcomes that will address United Nations Sustainable Development Goals 3 and 10 (good health and well being and reduced inequalities). In order to appropriately contextualize a curriculum using simulation, it is necessary to first conduct a needs assessment of the target learner population. The World Health Organization (WHO) has published a tool to assess capacity for emergency and surgical care in LMICs that is adaptable to this goal.

Materials And Methods: The WHO Tool for Situational Analysis to Assess Emergency and Essential Surgical Care was modified to assess pediatric resuscitation capacity in clinical settings in two LMICs: Uganda and Myanmar. Modifications included assessment of self-identified learning needs, current practices, and perceived epidemiology of disease burden in each clinical setting, in addition to assessment of pediatric resuscitation capacity in regard to infrastructure, procedures, equipment, and supplies. The modified tool was administered to 94 respondents from the two settings who were target learners of a proposed simulation-based curriculum in pediatric and neonatal resuscitation.

Results: Infectious diseases (respiratory illnesses and diarrheal disease) were cited as the most common causes of pediatric deaths in both countries. Self-identified learning needs included knowledge and skill development in pediatric airway/breathing topics, as well as general resuscitation topics such as CPR and fluid resuscitation in shock. Equipment and supply availability varied substantially between settings, and critical shortages were identified in each setting. Current practices and procedures were often limited by equipment availability or infrastructural considerations.

Discussion And Conclusion: Epidemiology of disease burden reported by respondents was relatively consistent with WHO country-specific UFMR statistics in each setting. Results of the needs assessment survey were subsequently used to refine goals and objectives for the simulation curriculum and to ensure delivery of pragmatic educational content with recommendations that were contextualized for local capacity and resource availability. Effective use of the tool in two different settings increases its potential generalizability.
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http://dx.doi.org/10.3389/fped.2018.00037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863499PMC
February 2018

Do End-of-Rotation and End-of-Shift Assessments Inform Clinical Competency Committees' (CCC) Decisions?

West J Emerg Med 2018 Jan 13;19(1):121-127. Epub 2017 Dec 13.

Johns Hopkins University Department of Emergency Medicine, Baltimore, Maryland.

Introduction: Clinical Competency Committees (CCC) require reliable, objective data to inform decisions regarding assignment of milestone proficiency levels, which must be reported to the Accreditation Council for Graduate Medical Education. After the development of two new assessment methods, the end-of-shift (EOS) assessment and the end-of-rotation (EOR) assessment, we sought to evaluate their performance. We report data on the concordance between these assessments, as well as how each informs the final proficiency level determined in biannual CCC meetings. We hypothesized that there would be a high concordance level between the two assessment methods, including concordance of both the EOS and EOR with the final proficiency level designation by the CCC.

Methods: The residency program is an urban academic four-year emergency medicine residency with 48 residents. After their shifts in the emergency department (ED), residents handed out EOS assessment forms asking about individual milestones from 15 subcompetencies to supervising physicians, as well as triggered electronic EOR-doctor (EORd) assessments to supervising doctors and EOR-nurse (EORn) to nurses they had worked with after each two-week ED block. EORd assessments contained the full proficiency level scale from 16 subcompetencies, while EORn assessments contained four subcompetencies. Data reports were generated after each six-month assessment period and data was aggregated. We calculated Spearman's rank order correlations for correlations between assessment types and between assessments and final CCC proficiency levels.

Results: Over 24 months, 5,234 assessments were completed. The strongest correlations with CCC proficiency levels were the EORd for the immediate six-month assessment period prior (r 0.71-0.84), and the CCC proficiency levels from the previous six-months (r 0.83-0.92). EOS assessments had weaker correlations (r 0.49 to 0.62), as did EORn (r 0.4 to 0.73).

Conclusion: End-of-rotation assessments completed by supervising doctors are most highly correlated with final CCC proficiency level designations, while end-of-shift assessments and end-of-rotation assessments by nurses did not correlate strongly with final CCC proficiency levels, both with overestimation of levels noted. Every level of proficiency the CCC assigned appears to be highly correlated with the designated level in the immediate six-month period, perhaps implying CCC members are biased by previous level assignments.
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http://dx.doi.org/10.5811/westjem.2017.10.35290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785178PMC
January 2018

Molecular analysis of high-grade serous ovarian carcinoma with and without associated serous tubal intra-epithelial carcinoma.

Nat Commun 2017 10 17;8(1):990. Epub 2017 Oct 17.

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Many high-grade serous carcinomas (HGSCs) of the pelvis are thought to originate in the distal portion of the fallopian tube. Serous tubal intra-epithelial carcinoma (STIC) lesions are the putative precursor to HGSC and identifiable in ~ 50% of advanced stage cases. To better understand the molecular etiology of HGSCs, we report a multi-center integrated genomic analysis of advanced stage tumors with and without STIC lesions and normal tissues. The most significant focal DNA SCNAs were shared between cases with and without STIC lesions. The RNA sequence and the miRNA data did not identify any clear separation between cases with and without STIC lesions. HGSCs had molecular profiles more similar to normal fallopian tube epithelium than ovarian surface epithelium or peritoneum. The data suggest that the molecular features of HGSCs with and without associated STIC lesions are mostly shared, indicating a common biologic origin, likely to be the distal fallopian tube among all cases.High-grade serous carcinomas (HGSCs) are associated with precursor lesions (STICs) in the fallopian epithelium in only half of the cases. Here the authors report the molecular analysis of HGSCs with and without associated STICs and show similar profiles supporting a common origin for all HGSCs.
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http://dx.doi.org/10.1038/s41467-017-01217-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645359PMC
October 2017

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.

Science 2017 07 8;357(6349):409-413. Epub 2017 Jun 8.

Bloomberg-Kimmel Institute for Cancer Immunotherapy at Johns Hopkins, Baltimore, MD 21287, USA.

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers' tissue of origin.
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http://dx.doi.org/10.1126/science.aan6733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576142PMC
July 2017

HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche.

Nat Commun 2017 04 28;8:15008. Epub 2017 Apr 28.

Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross Research Building, Room 1025, Baltimore, Maryland 21205, USA.

High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to 'build' an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis.
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http://dx.doi.org/10.1038/ncomms15008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414379PMC
April 2017

Transcriptional Mechanisms of Resistance to Anti-PD-1 Therapy.

Clin Cancer Res 2017 Jun 13;23(12):3168-3180. Epub 2017 Feb 13.

Department of Surgery, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland.

To explore factors associated with response and resistance to anti-PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response. Twenty-six melanoma specimens (four premortem, 22 postmortem) were subjected to whole exome sequencing. Candidate immunologic markers and gene expression were assessed in 10 cutaneous metastases showing response or progression during therapy. The melanoma was driven by biallelic inactivation of All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with overexpression of genes associated with extracellular matrix and neutrophil function. Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti-PD-1 outcomes. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474192PMC
June 2017

Optimizing the Use of Gene Expression Profiling in Early-Stage Breast Cancer.

J Clin Oncol 2016 12 31;34(36):4390-4397. Epub 2016 Oct 31.

Hyun-seok Kim, Christopher B. Umbricht, Peter B. Illei, Ashley Cimino-Mathews, Soonweng Cho, Nivedita Chowdhury, Maria Cristina Figueroa-Magalhaes, Catherine Pesce, Stacie C. Jeter, Deborah K. Armstrong, Roisin M. Connolly, John H. Fetting, Ben Ho Park, Vered Stearns, Antonio C. Wolff, and Leslie Cope, The Johns Hopkins University School of Medicine; Kala Visvanathan, The Johns Hopkins University Bloomberg School of Public Health, Baltimore; Charles Mylander, Martin Rosman, and Lorraine Tafra, Anne Arundel Medical Center, Annapolis, MD; Bradley M. Turner, David G. Hicks, University of Rochester Medical Center, Rochester, NY; Robert S. Miller, American Society of Clinical Oncology, Alexandria, VA; and Tyler A. Jensen and Dylan V. Miller, Intermountain Healthcare, Salt Lake City, UT.

Purpose Gene expression profiling assays are frequently used to guide adjuvant chemotherapy decisions in hormone receptor-positive, lymph node-negative breast cancer. We hypothesized that the clinical value of these new tools would be more fully realized when appropriately integrated with high-quality clinicopathologic data. Hence, we developed a model that uses routine pathologic parameters to estimate Oncotype DX recurrence score (ODX RS) and independently tested its ability to predict ODX RS in clinical samples. Patients and Methods We retrospectively reviewed ordered ODX RS and pathology reports from five institutions (n = 1,113) between 2006 and 2013. We used locally performed histopathologic markers (estrogen receptor, progesterone receptor, Ki-67, human epidermal growth factor receptor 2, and Elston grade) to develop models that predict RS-based risk categories. Ordering patterns at one site were evaluated under an integrated decision-making model incorporating clinical treatment guidelines, immunohistochemistry markers, and ODX. Final locked models were independently tested (n = 472). Results Distribution of RS was similar across sites and to reported clinical practice experience and stable over time. Histopathologic markers alone determined risk category with > 95% confidence in > 55% (616 of 1,113) of cases. Application of the integrated decision model to one site indicated that the frequency of testing would not have changed overall, although ordering patterns would have changed substantially with less testing of estimated clinical risk-high or clinical risk-low cases and more testing of clinical risk-intermediate cases. In the validation set, the model correctly predicted risk category in 52.5% (248 of 472). Conclusion The proposed model accurately predicts high- and low-risk RS categories (> 25 or ≤ 25) in a majority of cases. Integrating histopathologic and molecular information into the decision-making process allows refocusing the use of new molecular tools to cases with uncertain risk.
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http://dx.doi.org/10.1200/JCO.2016.67.7195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455310PMC
December 2016

Comparison of 20% sulfur hexafluoride with air for intraocular tamponade in Descemet membrane endothelial keratoplasty (DMEK).

Arq Bras Oftalmol 2016 Sep-Oct;79(5):299-302

Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD, United States.

Purpose:: To compare the effect of 20% sulfur hexafluoride (SF6) with that of air on graft detachment rates for intraocular tamponade in Descemet membrane endothelial keratoplasty (DMEK).

Methods:: Forty-two eyes of patients who underwent DMEK by a single surgeon (A.S.J.) at Wilmer Eye Institute between January 2012 and 2014 were identified; 21 received air for intraocular tamponade and the next consecutive 21 received SF6. The main outcome measure was the graft detachment rate; univariate and multivariate analyses were performed.

Results:: The graft detachment rate was 67% in the air group and 19% in the SF6 group (p<0.05). No complete graft detachments occurred, and all partial detachments underwent intervention with injection of intraocular air. The percentages of eyes with 20/25 or better vision were not different between the groups (67% vs. 71%). Univariate analysis showed significantly higher detachment rates with air tamponade (OR, 8.50; p<0.005) and larger donor graft size (OR, 14.96; p<0.05). Multivariate analysis with gas but not graft size included showed that gas was an independent statistically significant predictor of outcome (OR, 6.65; p<0.05). When graft size was included as a covariate, gas was no longer a statistically significant predictor of detachment but maintained OR of 7.81 (p=0.063) similar to the results of univariate and multivariate analyses without graft size.

Conclusion:: In comparison with air, graft detachment rates for intraocular tamponade in DMEK were significantly reduced by 20% SF6.
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http://dx.doi.org/10.5935/0004-2749.20160086DOI Listing
July 2017

Combination Epigenetic Therapy in Advanced Breast Cancer with 5-Azacitidine and Entinostat: A Phase II National Cancer Institute/Stand Up to Cancer Study.

Clin Cancer Res 2017 Jun 15;23(11):2691-2701. Epub 2016 Dec 15.

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland.

In breast cancer models, combination epigenetic therapy with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor led to reexpression of genes encoding important therapeutic targets, including the estrogen receptor (ER). We conducted a multicenter phase II study of 5-azacitidine and entinostat in women with advanced hormone-resistant or triple-negative breast cancer (TNBC). Patients received 5-azacitidine 40 mg/m (days 1-5, 8-10) and entinostat 7 mg (days 3, 10) on a 28-day cycle. Continuation of epigenetic therapy was offered with the addition of endocrine therapy at the time of progression [optional continuation (OC) phase]. Primary endpoint was objective response rate (ORR) in each cohort. We hypothesized that ORR would be ≥20% against null of 5% using Simon two-stage design. At least one response was required in 1 of 13 patients per cohort to continue accrual to 27 per cohort (type I error, 4%; power, 90%). There was one partial response among 27 women with hormone-resistant disease (ORR = 4%; 95% CI, 0-19), and none in 13 women with TNBC. One additional partial response was observed in the OC phase in the hormone-resistant cohort ( = 12). Mandatory tumor samples were obtained pre- and posttreatment (58% paired) with either up- or downregulation of ER observed in approximately 50% of posttreatment biopsies in the hormone-resistant, but not TNBC cohort. Combination epigenetic therapy was well tolerated, but our primary endpoint was not met. OC phase results suggest that some women benefit from epigenetic therapy and/or reintroduction of endocrine therapy beyond progression, but further study is needed. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457329PMC
June 2017

Monitoring of Serum DNA Methylation as an Early Independent Marker of Response and Survival in Metastatic Breast Cancer: TBCRC 005 Prospective Biomarker Study.

J Clin Oncol 2017 Mar 21;35(7):751-758. Epub 2016 Nov 21.

Kala Visvanathan, Johns Hopkins University School of Medicine and Bloomberg School of Public Health; MaryJo S. Fackler, Zhe Zhang, Zoila A. Lopez-Bujanda, Stacie C. Jeter, Lori J. Sokoll, Leslie M. Cope, Christopher B. Umbricht, David M. Euhus, Saraswati Sukumar, and Antonio C. Wolff, Johns Hopkins University School of Medicine, Baltimore, MD; Elizabeth Garrett-Mayer, Medical University of South Carolina, Charleston, SC; Andres Forero, University of Alabama at Birmingham, Birmingham, AL; Anna M. Storniolo, Indiana University, Bloomington, IN; Rita Nanda, University of Chicago, Chicago, IL; Nancy U. Lin, Dana-Farber Cancer Institute, Boston, MA; Lisa A. Carey, University of North Carolina, Chapel Hill, NC; and James N. Ingle, Mayo Clinic, Rochester, MN.

Purpose Epigenetic alterations measured in blood may help guide breast cancer treatment. The multisite prospective study TBCRC 005 was conducted to examine the ability of a novel panel of cell-free DNA methylation markers to predict survival outcomes in metastatic breast cancer (MBC) using a new quantitative multiplex assay (cMethDNA). Patients and Methods Ten genes were tested in duplicate serum samples from 141 women at baseline, at week 4, and at first restaging. A cumulative methylation index (CMI) was generated on the basis of six of the 10 genes tested. Methylation cut points were selected to maximize the log-rank statistic, and cross-validation was used to obtain unbiased point estimates. Logistic regression or Cox proportional hazard models were used to test associations between the CMI and progression-free survival (PFS), overall survival (OS), and disease status at first restaging. The added value of the CMI in predicting survival outcomes was evaluated and compared with circulating tumor cells (CellSearch). Results Median PFS and OS were significantly shorter in women with a high CMI (PFS, 2.1 months; OS, 12.3 months) versus a low CMI (PFS, 5.8 months; OS, 21.7 months). In multivariable models, among women with MBC, a high versus low CMI at week 4 was independently associated with worse PFS (hazard ratio, 1.79; 95% CI, 1.23 to 2.60; P = .002) and OS (hazard ratio, 1.75; 95% CI, 1.21 to 2.54; P = .003). An increase in the CMI from baseline to week 4 was associated with worse PFS ( P < .001) and progressive disease at first restaging ( P < .001). Week 4 CMI was a strong predictor of PFS, even in the presence of circulating tumor cells ( P = .004). Conclusion Methylation of this gene panel is a strong predictor of survival outcomes in MBC and may have clinical usefulness in risk stratification and disease monitoring.
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http://dx.doi.org/10.1200/JCO.2015.66.2080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455421PMC
March 2017