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2.0 Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Writer and Curator: Larry H Bernstein, MD, FCAP

This is the second article in a series concerning genomic expression, The first of which was concerned with the expanded technologies in use for study of genomic expression.  This portion will also cover more of genetic errors as well as methodologies, but not all examples are in the realm of cancer.

I shall start with a New York Times editorial on July 24, 2015 by Angelina Jolie Pitt on her experience with BRCA1 gene and her family history.  It is very instructive on how she worked through her experience.

http://www.nytimes.com/2015/03/24/opinion/angelina-jolie-pitt-diary-of-a-surgery.html?

Two years ago she was found to have a positive test for BRCA1, carrying an 87 percent risk for breast cancer and a 50 percent risk for ovarian cancer.  At that time she had a preventive mastectomy.  The decision was not easy, but it also brought into consideration that her mother and grandmother both died of breast cancer.  She did not have an oophorectomy at that time because on considering the advice of medical experts, she would have been left with no estrogen support. She wanted to delay her early vegetative senescence.  She has reached the age of 39 years and on the advice of medical expert opinion, she proceeded with salpingo-oophorectomy, at age 39 years, a decade before  her  mother had developed cancer.  But her delay was to allow her to recover and adjust emotionally to her ongoing situation, with a remaining risk for ovarian cancer.

She tested negative for CA-125^1-5 at this time prior to surgery. But the CA-125 test could well be negative with early onset ovarian cancer. It may be considered a better test for following treatment than for early diagnosis. Her choice was to sacrifice early menopause to the ability to live through her childrens’ childhood development.  This was a well thought out decision.  In addition, there were abnormal inflammatory markers that were not specific for cancer rsik, but were worth taking into account.  The procedure itself was simpler than the mastectomy.

http://static01.nyt.com/images/2015/03/23/opinion/23op-ed/23op-ed-master315.jpg

2.1  CA-125 and Ovarian Cancer

2.1.1  lmmunoradiometric Assay of CA 125 in Effusions: Comparison with Carcinoembryonic Antigen

Marguerite M. Pinto, MD,‘ Larry H. Bernstein, MD,* Dennis A. Brogan, MPH, MT

and Elaine Criscuolo, CT(ASCP) CMIACS

The levels of CA 125 antigen were measured in 167 effusions from 150 patients using radioimmunoassay, and the results compared with the levels of carcinoembryonic antigen (CEA) in the fluids. The results indicate that an elevated fluid CA 125 level (>14,000 U/ml-68,000 U/ml) and a negative fluid CEA level (4 ng/ml) is suggestive of serous and endometrioid carcinoma of ovary, and adenocarcinoma of the endometrium and fallopian tube. Alternatively, an elevated fluid CEA level (14 ng/ml-600 ng/ml) and a negative CA 125 level (20-5000 U/ml) is seen in metastatic carcinomas of breast, lung, gastrointestinal tract, and mucinous ystadenocarcinoma. Lymphomas, melanomas, and benign effusions are negative for both antigens. The combined use of CEA and CA 125 antigen in fluids is useful in the differential diagnosis of adenocarcinoma of unknown primary. Cancer 59:218-222, 1987.

2.1.2 CA-125 in fine-needle aspirates of solid tumors: comparison with cytologic diagnosis and carcinoembryonic antigen (CEA) assay.

Marguerite M. Pinto, S Kotta

Diagnostic Cytopathology 03/1996; 14(2):121-5.
http://dx.doi.org:/10.1002/(SICI)1097-0339(199603)14:2<121::AID-DC4>3.0.CO;2-M

One hundred and twenty-two fine needle aspirates (FNA) from female patients were studied to determine whether CA-125 assay contributed to cytologic diagnosis and CEA assay. Cytologic examination was done on Papanicolaou-stained smears and cell blocks, CEA by EIA (Abbott Laboratory, > 5 ng/ml cutoff) and CA-125 by RIA (Abbott Laboratory, North Chicago, IL, > 66 mu/ml cutoff). Final diagnosis were correlated with histologic diagnosis when available, clinical, radiologic studies, and follow-up. Results: 29 benign, 93 malignant. Sensitivities and specificities: cytology, 91%, 100%; CEA: 59%, 86%; CA-125, 50%, 55%. CEA plus cytology sensitivity, 97%. CA-125 content was highest in endometrial/ovarian carcinoma (39,899 mu/ml) and < 5,000 mu/ml in other tumors and benign FNA in contrast to CEA which showed highest levels in carcinomas of colon, pancreas, and lung (> 280 ng/ml). While elevated CEA enhances the sensitivity of cytologic diagnosis of carcinomas of the colon, pancreas, and lung, low CEA and high CA-125 content supports an ovarian/endometrial primary.

2.1.3  Diagnostic efficiency of carcinoembryonic antigen and CA125 in the cytological evaluation of effusions.

Pinto MM, Bernstein LH, Rudolph RA, Brogan DA, Rosman M.
Arch Pathol Lab Med. 1992 Jun; 116(6):626-31.

In our previous study, the combination of the concentrations of carcinoembryonic antigen (CEA) and CA125 and the findings from cytological examination in 189 benign and malignant pleural and peritoneal effusions was useful in the diagnosis/classification of malignant effusions. Sensitivity of CEA (level, greater than 5 ng/mL) was 68%; specificity was 99% for the diagnosis of malignant effusions secondary to carcinoma of the lung, breast, gastrointestinal tract, and mucinous carcinoma of the ovary. Sensitivity of CA125 (level, greater than 5000 U/mL) was 85%; specificity was 96% for the diagnosis of malignant effusions in carcinoma of the ovary, fallopian tube, and endometrium. We now expanded the study to include 840 pleural and peritoneal effusions (benign, n = 520; malignant, n = 320) and analyzed the data by the statistical method of Rudolph and colleagues. Based on new cutoff values, ie, CEA level at 6.3 ng/mL and CA125 level at 3652 U/mL, the sensitivities for detection of malignant effusions secondary to carcinomas of the lung, breast, and gastrointestinal tract and mucinous carcinoma of the ovary varied between 75% and 100%; specificity was 98%. Sensitivity of CA125 for detection of malignant effusions from müllerian epithelial carcinoma was 71%; specificity was 99%. The elevated CEA fluid level alone helped to diagnose malignant effusions of the gastrointestinal tract in 54%, breast in 19%, and lung in 16%. The high CA125 fluid level was predictive of müllerian epithelial carcinoma. Adjunctive use of CEA and CA125 levels in fluid enhances the sensitivity of cytological diagnosis and may be predictive of the primary site in patients who present with carcinoma of an unknown primary source.

2.2 Carcinoembryonic antigen in diagnostics

2.2.1 Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Pinto MM1, Ha DJ.
Acta Cytol. 1992 May-Jun; 36(3):277-82

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.

2.2.2  Relationship between serum CA125 half life and survival in ovarian cancer

Table
Gupta and Lis Journal of Ovarian Research 2009 2:13
http://dx.doi.org:/10.1186/1757-2215-2-13

First Author, Year, Study Place	Data Collection	Study Design	Sample Size	RR/HR, (95% CI), P-Value
Riedinger JM, 2006, France	1988 to 1996	R	553	2.04 (1.58-2.63), < 0.0001
Gadducci A, 2004, Italy	1996 to2002	R	71	3.11 (1.22-7.98), 0.0181
Munstedt K, 1997, Germany	1987 to1994	R	85	0.6184
Gadducci A, 1995, Italy	1986 to1992	R	225	2.13 (1.23-3.68), 0.0073
Rosman M, 1994, Connecticut	1985 to 1989	R	51	3.6 (1.8-7.4), < 0.001
Yedema C A, 1993, Netherlands	1984 to 1990	R	60	9.17 (1.49-56.3), 0.01
Hawkins RE, 1989, London	NA	P	29	3.7 (0.7-20.1), 0.001;27.8 (4.0-193), 0.001

¹CA125 half-life was independent prognostic indicator for survival
²FIGO stage, tumor grade, residual disease, CA125
http://www.ovarianresearch.com/content/2/1/13/table/T6

3.3.0      DNA double strand breaks

2.3.1.  Collaboration and competition – DNA double-strand break repair pathways

Kass EM, Jasin M
FEBS Letters 2010; 584:3703-3708
http://dx.doi.org:/jfebslet.2010.07.057

DNA double-strand breaks occur in replication and exogenous sources pose risk to genome stability. There are two pathways to repair.  They are non-homologous end joining and homologous recombination. Both pathways cooperate and compete at double-strand break sites.

2.3.2 DNA Double-Strand Break Repair Inhibitors as Cancer Therapeutics

Srivastava M, Rashavan SC
Chem & Biol 2015 Jan; pp17-29
http://dx.doi.org:/10.1016/jchembiol.2014.11.013

Homologous recombination and non-homologous end joining are the two major repair pathways expressed in eukaryotes.  For double-strand breaks, and the DSB repair gene is vulnerable to chemotherapy and radiation therapy, accounting for treatment resistance. Therefore, targeting DSB repair is attractive. Blocking the residual repair using inhibitors can potentiate treatment.

2.3.3  Animation published in DNA Repair: Helleday T, Lo J, van Gent DC, Engelward BP. DNA double-strand break repair: From mechanistic understanding to cancer treatment. DNA Repair. (14 Mar 2007)
2.3.3.1 http://web.mit.edu/engelward-lab/animations/DSBR.html

2.3.3.2 https://www.youtube.com/watch?v=eg8rpYFsqCA

2.3.4 Homology-dependent double strand break repair. Oxford Academic (Oxford University Press)

https://www.youtube.com/watch?v=86JCMM5kb2A

2.4.0 Managing DNA data sets

2.4.1 Bionimbus –  a cloud for managing, analyzing and sharing large genomics datasets

The Bionimbus Protected Data Cloud (PDC) is a collaboration between the Open Science Data Cloud (OSDC) and the IGSB (IGSB,) the Center for Research Informatics (CRI), the Institute for Translational Medicine (ITM), and the University of Chicago Comprehensive Cancer Center (UCCCC). The PDC allows users authorized by NIH to compute over human genomic data from dbGaP in a secure compliant fashion. Currently, selected datasets from the The Cancer Genome Atlas (TCGA) are available in the PDC.

https://bionimbus-pdc.opensciencedatacloud.org/

2.4.1.2 Accounting for uncertainty in DNA sequencing data

O’Rawe JA, Ferson S, Lyon GJ
Trends in Genetics 2015 Feb; 31(2):61-66
http://dx.doi.org:/10.101/jtig.2014.12.002

This article reviews uncertainty in quantification in DNA sequency applications and sources of error propagation, and it proposes methods to account for errors and uncertainties.

2.5.0 Linking Traits to Mechanisms and UPR response/proteostasis

2.5.1 Stress-Independent Activation of XBP1s and/or ATF6 Reveals –Three Linking traits based on their shared molecular mechanisms

Shoulders MD, Ryno LM, Genereux JC,…Wiseman BL
Cell Reports 2013 Apr; 3, pp 1279-1292
http://dx.doi.org:/10.1016/j.celrep.2013.03.024

The unfolded protein response (UPR) maintains ER proteostasis through the transcription factors XP1s and ATF6. This study measured orthogonal small molecule-mediated activation of transcription factors nXP1s and/or ATF6 using transcriptomics and quantitative proteomics. The finding is that three ER proteostasis environmants differentially influence

1. Folding
2. Traffiking, and
3. Degradation of destabilized ER client proteins

Without affecting endogenous proteome. The proteostasis network is remodeled with the potential for selective restoration of the aberrant ER proteostasis.

2.5.2 Biological and chemical approaches to diseases of proteostasis deficiency.

Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE
Annu Rev Biochem. 2009; 78:959-91.
http://dx.doi.org:/10.1146/annurev.biochem.052308.114844

Many diseases appear to be caused by the misregulation of protein maintenance. Such diseases of protein homeostasis, or “proteostasis,” include loss-of-function diseases (cystic fibrosis) and gain-of-toxic-function diseases (Alzheimer’s, Parkinson’s, and Huntington’s disease). Proteostasis is maintained by the proteostasis network, which comprises pathways that control protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. The decreased ability of the proteostasis network to cope with inherited misfolding-prone proteins, aging, and/or metabolic/environmental stress appears to trigger or exacerbate proteostasis diseases. Herein, we review recent evidence supporting the principle that proteostasis is influenced both by an adjustable proteostasis network capacity and protein folding energetics, which together determine the balance between folding efficiency, misfolding, protein degradation, and aggregation. We review how small molecules can enhance proteostasis by binding to and stabilizing specific proteins (pharmacologic chaperones) or by increasing the proteostasis network capacity (proteostasis regulators). We propose that such therapeutic strategies, including combination therapies, represent a new approach for treating a range of diverse human maladies.

2.5.3 Extracellular Chaperones and Proteostasis

Amy R. Wyatt, Justin J. Yerbury, Heath Ecroyd, and Mark R. Wilson
Annual Review of Biochemistry 2013 Jun; 82: 295-322
http://dx.doi.org:/10.1146/annurev-biochem-072711-163904

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world’s most debilitating, costly, and intractable diseases.

http://www.proteostasis.com/our-technology/proteostasis-network.html

http://www.proteostasis.com/images/stories/technology/illustration1.gif

2.6.0 Transcription

2.6.1 Looping Back to Leap Forward. Transcription Enters a New Era

Levine M, Cattoglio C, Tijan R
Cell 2014 Mar; 157: 13-22.
http://dx.doi.org:/10.1016/j.cell.2014.02.009

Organism complexity is not in gene number, but lies in gene regulation. The human genbome contains hundreds of thousands of enhancers, and genes are embedded in a milieu of enhancers . Proliferation of cis-regulatory DNAs is accompanied by complexity and functional diversity of transcription machinery recognizing distal enhancers and promotors, and high-order spatial organization. This article reviews the dynamic communication of remote enhancers with target promoters.

2.6.2 Activating gene expression in mammalian cells with promoter-targeted duplex RNAs.

Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR.
Nat Chem Biol. 2007 Mar; 3(3):166-73
http://dx.doi.org:/10.1038/nchembio860

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.
2.6.3 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

M Gossen and H Bujard
Proc Natl Acad Sci U S A. 1992 Jun 15; 89(12): 5547–5551.

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of “on/off” situations for such genes in a reversible way.

http://www.intechopen.com/source/html/16788/media/image5.jpeg

http://openi.nlm.nih.gov/imgs/512/321/2408727/2408727_pone.0002357.g001.png

2.7.0 Epigenetics and Cancer

2.7.1 Epigenetics and cancer metabolism

Johnson C, Warmoes MO, Shen X, Locasale JW
Cancer Letters 2015;  356:309-314.
http://dx.doi.org:/10.1016/j.canlet.2013.09.043

Cancer is characterized by adaptive metabolic changes for proliferation and survival of the neoplastic cell, which is accompanied by dysfunctional metabolic enzyme changes in a specific nutrient supplied environment. The oncogenic change uses epigenetic level enzymes that catalyze posttranslational modifications of the DNA/histone expression with metabolites including cofactors and substrates for reactions. This interaction of epigenetics and metabolism provides new insights for anti-cancer therapy.

2.7.2 Cancer Epigenetics. From Mechanism to Therapy

Dawson MA, Konzarides T
Cell 2012 Jul; 150:12-27
http://dx.doi.org:/10.1016/j.cell.2012.06.013

Carcinogenesis requires all of the following:

• DNA methylation
• Histone modification
• Nucleosome remodeling
• RNA mediated targeting

This article reviews basic principles of epigenetic pathways that are dysregulated in carcinogenesis.

2.7.4 A subway review of cancer pathways

Hahn WC, Weinberg RA
Nature Reviews: Cancer
http://www.nature.com/nrc/poster/subpathways/index.html

Cancer arises from the stepwise accumulation of genetic changes that confer upon an incipient neoplastic cell the properties of unlimited, self-sufficient growth and resistance to normal homeostatic regulatory mechanisms. Advances in human genetics and molecular and cellular biology have identified a collection of cell phenotypes � the main destinations in the subway map below � that are required for malignant transformation1. Specific molecular pathways (subway lines) are responsible for programming these behaviours. Although the connections between cancer-cell wiring and function remain incompletely explored and specified � hence the many lines under construction � the broad outlines of the molecular circuitry of the cancer cell can now be sketched. Further advances in understanding these pathways and their interconnections will accelerate the development of molecularly targeted therapies that promise to change the practice of oncology.

http://www.nature.com/nrc/poster/subpathways/images/map.gif

Subway map designed by Claudia Bentley.