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Ethical Concerns in Personalized Medicine: BRCA1/2 Testing in Minors and Communication of Breast Cancer Risk

Reporter/Curator: Stephen J. Williams, Ph.D.

Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing[1]

Direct-to-consumer (DTC) genetic testing and genetic health information in 2007 with the advent of personalized testing services by companies who offered microarray-based genotyping of single-nucleotide-polymorphisms (SNP) which had strong correlations to disease risk.  Three companies started to offer such services directly to the consumer:

A common test which is offered analyzes the consumers BRCA1/2 mutation status.  Three mutations in the BRCA gene are known to predispose women to hereditary breast and ovarian cancer: BRCA1 185delAG, BRCA1 538insC, and BRCA2 617delT.  These BRCA1 mutation confer a 60% breast cancer risk and a 40% risk of ovarian cancer while the BRCA2 mutation confers a breast cancer risk of 50% and 20% risk of ovarian cancer.

However, the commercial availability of this genetic disease-risk associated testing has led to certain ethical issues concerning communication and responses of risk information by:

  1. Consumers who request BRCA1/2 testing (focus of the Francke article
  2. BRCA1/1 testing and communication of results to minors and relatives (Bradbury: see below)

There has been much opinion, either as commentary in literature, meeting proceedings, or communiques from professional societies warning that this type of “high-impact” genetic information should not be given directly to the consumer as consumers will not fully understand the information presented to them, be unable to make proper risk-based decisions, results could cause panic and inappropriate action such as prophylactic oophorectomy or unwarranted risk-reduction mastectomy, or false reassurance in case of negative result and reduced future cancer screening measures taken by the consumer.  However, there have been few studies to investigate these concerns.

A report by Dr. Uta Francke in the open access journal PeerJ, assesses and quantifies the emotional and behavioral reactions of consumers to their 23andMe Personal Genome Service® report of the three BRCA mutations known to be associated with high risk for breast/ovarian cancer.  One hundred thirty six (136) individuals, who tested positive for BRCA1 and/or BRCA2 mutations as well as 160 users of the service, who tested mutation-free were invited to participate in phone interviews addressing personal and family history of cancer, decision and timing of viewing the BRCA report, recollection of results, emotional responses, perception of personal cancer risk, information sharing, and actions taken.  Thirty two (32) mutation carriers (16 female and 16 male) and 31 non-carriers responded to the phone questionnaire.

Questions were based on the following themes:

  1. When you purchased the 23andME Personal Genome Service® were you aware that it included testing for mutations that predispose to breast and ovarian cancer?
  2. Were you aware that having Ashkenazi Jewish ancestry influences your risk of carrying one of the three mutations?
  3. Have you or a first or second degree relative been diagnosed with breast, ovarian or any other cancer?
  4. What did you learn from your results?
  5. Were you surprised by the result?
  6. How did you feel about this information (extremely, moderately, somewhat upset or extremely relieved)?

Results:  Eleven women and 14 men had received an unexpected result that they are carriers of one of the three mutations however none of them reported extreme anxiety and only four reported moderate anxiety which did not last long.  Participants were at least 8 years of age. Five women and six men described their reaction as neutral.  Most carrier women sought medical advice and four underwent risk-reducing procedures. Some to the male carriers felt burdened to share their test results with their female relatives, which led to additional screenings of relatives.  Almost all of the mutation-positive customers appreciated learning their BRCA mutational status.

Other highlights of the results include:

  • More women got tested if they had a first or second degree relative previously diagnosed with breast/ovarian cancer
  • Ten mutation-positive individuals who were surprised at the test results cited the lack of family history of breast/ovarian cancer as the reason for their surprise.  The rest who were surprised at their positive test results believed that the frequency of these mutations were low in the general population so they shouldn’t have been affected.
  • For the mutation-positive group, none of the 32 reported as being “extremely upset”.
  • Interestingly, on male who learned, for the first time, he was a positive carrier for BRCA mutation, reported feeling “relieved” because his daughter who was also tested by 23andMe had not acquired his mutation.

A brief interview with Dr. Francke follows:

Q:     In your results you had noted that none of the mutation carriers showed extreme anxiety about their reports however there were many of Ashkenazi descent who was well aware of the increased risk to breast cancer.  In another study by Dr. Angela Bradbury, anxieties and communication to their children depended on mutation status and education status.  Do you feel that most women in your study were initially aware they could be in a high risk category for cancer, whether breast, ovarian, or other?

Dr. Francke:   As we show in Table 1, 6 of 16 women and 6 of 16 men who found out that they were BRCA mutation carriers had not been aware that being of Ashkenazi descent confers an increased risk of breast/ovarian cancer.  In Table S1, we show that 6 of these 32 people did not self-identify as Ashkenazi.
Q:     The reporting and communication of test results to offspring and genetic testing of offspring as a result of positive tests has been under much debate.  I had noticed that there was a high proportion of relatives who went for screening after learning of a family members BRCA testing, whether it showed a mutation or not.  Some studies have shown that offspring of carriers may misinterpret genetic testing results and take inappropriate action, such as considering having early testing  before age 25.  It appears some anxiety may be due to misinformation and lack of genetic counseling.  Should these test results be considered in guidelines for oncologist such as NCCN guidelines with respect to informing family members using genetic counselors as an intermediary?

Dr. Francke:    The “high proportion of relatives who went for screening after learning of a family member’s BRCA testing”, were only those related to a BRCA-positive person. Most of the BRCA testing of relatives was done through health care providers at Myriad as these people were eligible for insurance coverage of the test. In our interviews we found no evidence for inappropriate action of carriers or non-carriers. With one exception, we found no evidence for misinterpretation or “anxiety due to lack of genetic counseling”.  In our online reports we recommend genetic counseling for all customers who have questions about their results.

Q:     I was also particularly interested the male carrier felt a heightened burden to tell their offspring.  This has been suggested in other studies.  I would assume the mothers and not the fathers would feet more pressure to tell their children.  Is there a reason for this?
Dr. Francke:     The heightened burden reported by the male carriers was mostly about the realization of the risk for their daughters, not so much about to telling their offspring.  Female carriers were primarily concerned about their own health risk and management, and decision-making about preventive measures – therefore, the risk for offspring appeared to be of secondary concern for them.

However, the availability of this type of predictive genetic testing for hereditary cancer has raised some ethical issues regarding the communication of risk and genetic results to family members and especially offspring, specifically whether informing minors would incur unnecessary testing, anxiety among minors of parents who tested positive for genetic risk-factors, or even premature risk-reduction surgeries or medical interventions.

The aforementioned ethical issues concerning communicating results of BRCA mutational testing to offspring was addressed by two large studies conducted by Dr. Angela Bradbury M.D. and colleagues at Fox Chase Cancer Center Family Risk Assessment Program (now she is at University of Pennsylvania) and University of Chicago Cancer Risk Clinic.  These studies evaluated the parental opinions regarding BRCA1/2 testing of minors, and how parents communicate BRCA1/2 genetic testing with their children.

In the JCO article (Parent Opinions Regarding the Genetic Testing of Minors for BRCA1/2)[2], Bradbury and colleagues used semistructured interviews (yes/no questions and open-ended questions) of 246 parents at Fox Chase and University of Chicago, who underwent BRCA1/2 whether they supported testing of minors in general and testing of their own offspring.  Parents were asked, “If you were deciding, do you think children under 18 years old should be given the opportunity to be tested” and followed by the open-ended question: “Why do (don’t) you support the genetic testing of minors for BRCA1/2?”.

Results:  In response to the first question (Would you support testing in minors) 37% of parents supported testing of minors in the general population.  The follow-up open-ended question revealed that 4% support testing minors in some or all circumstances.  This decision was independent of parent sex or race.  44% of parents would test their own offspring.  Parents who opposed testing in minors thought testing would cause fear and anxiety for their children but those who supported unconditional testing (regardless of whether they were positive for the BRCA mutation or not) mentioned that the medical information would foster better health behaviors in their offspring.  21% of parents who opposed testing minors, in general, actually supported testing of their own children.  Interestingly parents who tested positive for the BRCA1 overwhelmingly (64%) opposed testing of minors, in general.  In addition, statistical analysis of the open-ended questions revealed that parents who did not have a college degree, had a negative test result, and were non white favored testing of their own children.  The authors had suggested larger studies before any guidelines were given as to whether testing in minors of BRCA mutation carriers should be standard.

In a recent publication by Dr. Bradbury and colleagues (Knowledge and perceptions of familial and genetic risks for breast cancer risk in adolescent girls)[3],  studied how adolescent girls understood and responded to breast cancer risk by interviewing 11-19 year-old girls at high-risk and population-risk for breast cancer. Although most girls said they were aware of increased risk because either a family member had or was predisposed to breast cancer (66 %) only 17 % of girls were aware of BRCA1/2 genes. Mother was the most frequently reported source of information for breast cancer among both high-risk (97 %) and population-risk (89 %) girls.  The study also showed that most girls who believe they are at high-risk could alter their lifestyles or change dietary habits to lower their risk.

In an adjacent study in the journal Cancer[4], Bradbury and colleagues at Fox Chase Cancer Center had gauged the frequency with which parents had told their children of their BRCA1/2 teat results and how their children felt about the results.

When parents disclose BRCA1/2 test results: Their communication and perceptions of offspring response[4]

Semi-structured interviews were conducted with parents who had BRCA1/2 testing and at least 1 child <25 YO.  A total of 253 parents completed interviews (61% response rate), reporting on 505 offspring. Twenty-nine percent of parents were BRCA1/2 mutation carriers. Three hundred thirty-four (66%) offspring learned of their parent’s test result. Older offspring age (P ≤ .01), offspring gender (female, P = .05), parents’ negative test result (P = .03), and parents’ education (high school only, P = .02) were associated with communication to offspring. The most frequently reported initial offspring responses were neutral (41%) or relief (28%). Thirteen percent of offspring were reported to experience concern or distress (11%) in response to parental communication of their test results. Distress was more frequently perceived among offspring learning of their parent’s BRCA1/2 positive or variant of uncertain significance result.

CONCLUSIONS:

Many parents communicate their BRCA1/2 test results to young offspring. Parents’ perceptions of offspring responses appear to vary by offspring age and parent test result. A better understanding of how young offspring respond to information about hereditary risk for adult cancer could provide opportunities to optimize adaptive psychosocial responses to risk information and performance of health behaviors, in adolescence and throughout an at-risk life span.

Below is an excellent article by Steven Reinberg from HealthDay interviewing Dr. Angela Bradbury concerning their JCO study: (reported for ABC News at http://abcnews.go.com/Health/Healthday/story?id=4508346&page=1#.UVNJUVef2RM)

Many Parents Share Genetic Test Findings With Kids

By Steven Reinberg
HealthDay Reporter

Mar. 23

FRIDAY, Aug. 17 (HealthDay News) — As genetic testing for diseases becomes more commonplace, the impact of those findings on family members may be underestimated, researchers say.

For instance, some women who discover they have the BRCA gene mutation, which puts them at higher risk for breast cancer, choose to tell their children about it before the children are old enough to understand the significance or deal with it, a new study found.

“Parents with the BRCA mutation are discussing their genetic test results with their offspring often many years before the offspring would need to do anything,” said study author Dr. Angela Bradbury, director of the Fox Chase Cancer Center’s Family Risk Assessment Program, in Philadelphia.

According to Bradbury, more than half of parents she surveyed told their children about genetic test results. Some parents reported that their children didn’t seem to understand the significance of the information, and some had initial negative reactions to the news.

“A lot of genetic information is being shared within families and there hasn’t been a lot of guidance from health-care professionals,” Bradbury said. “While this genetic risk may be shared accurately, there is risk of inaccurate sharing.”

In the study, Bradbury’s team interviewed 42 women who had the BRCA mutation. The researchers found that 55 percent of parents discussed the finding and the risk of breast cancer with at least one of their children who was under 25.

Also, most of the women didn’t avail themselves of the services of a doctor or genetic counselor in helping to tell their children, Bradbury’s group found.

Bradbury is concerned that sharing genetic information with young children can create anxiety. “The children could be overly concerned about their own risk at a time when there is nothing that they need to do,” she said.

But, she added, “it may be possible that sharing may be good for children in adapting to this information.”

The findings are published in the Aug. 20 issue of the Journal of Clinical Oncology.

The lack of definitive data on when — or if — to discuss genetic test results with children is a real problem, Bradbury said.

“As we move genetic testing forward for cancer or other illnesses, we have to consider the context of the whole family and focus our counseling to the whole family, and not just the person who comes in for testing,” Bradbury said. “We should learn more about how and when we should talk to children about this, so that we can promote healthy behaviors without causing too much anxiety for the offspring.”

Barbara Brenner, executive director of Breast Cancer Action, agreed that the psychological component of genetic testing needs more attention.

“This is the tip of a very scary iceberg,” Brenner said. “We don’t know the psychological consequences [of BRCA testing], not only to the person who has the test, but to her family members.”

Brenner thinks guidelines to help parents deal with this information are needed. So is help from doctors and genetic counselors in counseling family members, especially children, she added.

LEGACY (Lessons in Epidemiology and Genetics of Adult Cancer from Youth), supported by the National Institutes of Health. This study will follow the girls prospectively in order to evaluate epidemiologic and epigenetic pathways of childhood exposures in relation to pubertal development, age at menarche, breast tissue characteristics, biomarkers of exposure, genomic DNA methylation, and the psychosocial impact of increased breast cancer susceptibility in 6-13 YO girls. http://legacygirlsstudy.org/

1.         Francke U, Difamco C, Kiefer AK, Eriksson N, Moiseff B, Tung JY, Mountain JL: Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing. PeerJ 2013:1-21.

2.         Bradbury AR, Patrick-Miller L, Egleston B, Sands CB, Li T, Schmidheiser H, Feigon M, Ibe CN, Hlubocky FJ, Hope K et al: Parent opinions regarding the genetic testing of minors for BRCA1/2. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2010, 28(21):3498-3505.

3.         Bradbury AR, Patrick-Miller L, Egleston BL, Schwartz LA, Sands CB, Shorter R, Moore CW, Tuchman L, Rauch P, Malhotra S et al: Knowledge and perceptions of familial and genetic risks for breast cancer risk in adolescent girls. Breast cancer research and treatment 2012, 136(3):749-757.

4.         Bradbury AR, Patrick-Miller L, Egleston BL, Olopade OI, Daly MB, Moore CW, Sands CB, Schmidheiser H, Kondamudi PK, Feigon M et al: When parents disclose BRCA1/2 test results: their communication and perceptions of offspring response. Cancer 2012, 118(13):3417-3425.

Sources:

http://abcnews.go.com/Health/Healthday/story?id=4508346&page=1#.UVNJUVef2RM

Other article on Ethics and Personalized Medicine on the site include:

Genomics in Medicine- Tomorrow’s Promise

Attitudes of Patients about Personalized Medicine

Genomics & Ethics: DNA Fragments are Products of Nature or Patentable Genes?

Volume One: Genomics Orientations for Individualized Medicine

Directions for Genomics in Personalized Medicine

The Way With Personalized Medicine: Reporters’ Voice at the 8th Annual Personalized Medicine Conference,11/28-29, 2012, Harvard Medical School, Boston, MA

Highlights from 8th Annual Personalized Medicine Conference, November 28-29, 2012, Harvard Medical School, Boston, MA

Clinical Genetics, Personalized Medicine, Molecular Diagnostics, Consumer-targeted DNA – Consumer Genetics Conference (CGC) – October 3-5, 2012, Seaport Hotel, Boston, MA

Genetic basis of Complex Human Diseases: Dan Koboldt’s Advice to Next-Generation Sequencing Neophytes

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Improving Mammography-based imaging for better treatment planning

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From Molecular Biology to Translational Medicine: How Far Have We Come, and Where Does It Lead Us?

The Initiation and Growth of Molecular Biology and Genomics, Part I

Curator: Larry H Bernstein, MD, FCAP

Introduction and purpose

This material will cover the initiation phase of molecular biology, Part I; to be followed by the Human Genome Project, Part II; and concludes with Ubiquitin, it’s Role in Signaling and Regulatory Control, Part III.
This article is first a continuation of a previous discussion on the role of genomics in discovery of therapeutic targets titled Directions for genomics in personalized medicine https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

The previous article focused on key drivers of cellular proliferation, stepwise mutational changes coinciding with cancer progression, and potential therapeutic targets for reversal of the process. It also covers the race to delineation of the Human Genome, discovery methods and fundamental genomic patterns that are ancient in both animal and plant speciation.

This article reviews the web-like connections between early and later discoveries, as significant finding has led to novel hypotheses and many more findings over the last 75 years. This largely post WWII revolution has driven our understanding of biological and medical processes at an exponential pace owing to successive discoveries of chemical structure, the basic building blocks of DNA and proteins, of nucleotide and protein-protein interactions, protein folding, allostericity, genomic structure, DNA replication, nuclear polyribosome interaction, and metabolic control. In addition, the emergence of methods for copying, removal and insertion, and improvements in structural analysis as well as developments in applied mathematics have transformed the research framework.

In the Beginning

During the Second World War we had the discoveries of physics and the emergence out of the Manhattan Project of radioactive nuclear probes from E.O. Lawrence University of California Berkeley Laboratory. The use of radioactive isotopes led to the development of biochemistry and isolation of nucleotides, nucleosides, enzymes, and filling in of details of pathways for photosynthesis, for biosynthesis, and for catabolism.
Perhaps a good start of the journey is a student of Neils Bohr named Max Delbruck (September 4, 1906 – March 9, 1981), who won the Nobel prize for discovering that bacteria become resistant to viruses (phages) as a result of genetic mutations, founded a new discipline called Molecular Biology, lifting the experimental work in Physiology to a systematic experimentation in biology with the rigor of Physics using radiation and virus probes on selected cells. In 1937 he turned to research on the genetics of Drosophila melanogaster at Caltech, and two years later he coauthored a paper, “The growth of bacteriophage”, reporting that the viruses replicate in one step, not exponentially. In 1942, he and Salvador Luria of Indiana University demonstrated that bacterial resistance to virus infection is mediated by random mutation. This research, known as the Luria-Delbrück experiment, notably applied mathematics to make quantitative predictions, and earned them the 1969 Nobel Prize in Physiology or Medicine, shared with Alfred Hershey. His inferences on genes’ susceptibility to mutation was relied on by physicist Erwin Schrödinger in his 1944 book, What Is Life?, which conjectured genes were an “aperiodic crystal” storing code-script and influenced Francis Crick and James D. Watson in their 1953 identification of cellular DNA’s molecular structure as a double helix.

Watson-Crick Double Helix Model

A new understanding of heredity and hereditary disease was possible once it was determined that DNA consists of two chains twisted around each other, or double helixes, of alternating phosphate and sugar groups, and that the two chains are held together by hydrogen bonds between pairs of organic bases—adenine (A) with thymine (T), and guanine (G) with cytosine (C). Modern biotechnology also has its basis in the structural knowledge of DNA—in this case the scientist’s ability to modify the DNA of host cells that will then produce a desired product, for example, insulin.
The background for the work of the four scientists was formed by several scientific breakthroughs:

  1. the progress made by X-ray crystallographers in studying organic macromolecules;
  2. the growing evidence supplied by geneticists that it was DNA, not protein, in chromosomes that was responsible for heredity;
  3. Erwin Chargaff’s experimental finding that there are equal numbers of A and T bases and of G and C bases in DNA;
  4. and Linus Pauling’s discovery that the molecules of some proteins have helical shapes.

In 1962 James Watson (b. 1928), Francis Crick (1916–2004), and Maurice Wilkins (1916–2004) jointly received the Nobel Prize in physiology or medicine for their 1953 determination of the structure of deoxyribonucleic acid (DNA), performed with a knowledge of Chargaff’s ratios of the bases in DNA and having  access to the X-ray crystallography of Maurice Wilkins and Rosalind Franklin at King’s College London. Because the Nobel Prize can be awarded only to the living, Wilkins’s colleague Rosalind Franklin (1920–1958), who died of cancer at the age of 37, could not be honored.
Of the four DNA researchers, only Rosalind Franklin had any degrees in chemistry. Franklin completed her degree in 1941 in the middle of World War II and undertook graduate work at Cambridge with Ronald Norrish, a future Nobel Prize winner. She returning to Cambridge after a year of war service, presented her work and received the PhD in physical chemistry. Franklin then learned the  X-ray crystallography in Paris and rapidly became a respected authority in this field. Returning to returned to England to King’s College London in 1951, her charge was to upgrade the X-ray crystallographic laboratory there for work with DNA.

bt2304  Rosalind Franklin, crystallographer

Cold Spring Harbor Laboratory

I digress to the beginnings of the Cold Spring Harbor Laboratory. A significant part of the Laboratory’s life revolved around education with its three-week-long Phage Course, taught first in 1945 by Max Delbruck, the German-born, theoretical-physicist-turned-biologist. James D Watson first came to Cold Spring Harbor Laboratory with his thesis advisor, Salvador Luria, in the summer of 1948. Over its more than 25-year history, the Phage Course was the training ground for many notable scientists. The Laboratory’s annual scientific Symposium, has provided a unique highly interactive education about the exciting field of “molecular” biology. The 1953 symposium featured Watson coming from England to give the first public presentation of the DNA double helix. When he became the Laboratory’s director in 1968 he was determined to make the Laboratory an important center for advancing molecular biology, and he focused his energy on bringing large donations to the enterprise CSHNL. It became a magnate for future discovery at which James D. Watson became the  Director in 1968, and later the Chancellor. This contribution has as great an importance as his Nobel Prize discovery.

Biochemistry and Molecular Probes comes into View

Moreover, at the same time, the experience of Nathan Kaplan and Martin Kamen at Berkeley working with radioactive probes was the beginning of an establishment of Lawrence-Livermore Laboratories role in metabolic studies, as reported in the previous paper. A collaboration between Sid Collowick, NO Kaplan and Elizabeth Neufeld at the McCollum Pratt Institute led to the transferase reaction between the two main pyridine nucleotides.  Neufeld received a PhD a few years later from the University of California, Berkeley, under William Zev Hassid for research on nucleotides and complex carbohydrates, and did postdoctoral studies on non-protein sulfhydryl compounds in mitosis. Her later work at the NIAMDG on mucopolysaccharidoses. The Lysosomal Storage Diseases opened a new chapter on human genetic diseases when she found that the defects in Hurler and Hunter syndromes were due to decreased degradation of the mucopolysaccharides. When an assay became available for α-L-iduronidase in 1972, Neufeld was able to show that the corrective factor for Hurler syndrome that accelerates degradation of stored sulfated mucopolysaccharides was α-L-iduronidase.

______________________________________________________

The Hurler Corrective Factor. Purification and Some Properties (Barton, R. W., and Neufeld, E. F. (1971) J. Biol. Chem. 246, 7773–7779)
The Sanfilippo A Corrective Factor. Purification and Mode of Action (Kresse, H., and Neufeld, E. F. (1972) J. Biol. Chem. 247, 2164–2170)
_______________________________________________________

I mention this for two reasons:
[1] We see a huge impetus for nucleic acids and nucleotides research growing in the 1950’s with a post WWII emergence of work on biological structure.
[2] At the same time, the importance of enzymes in cellular metabolic processes runs parallel to that of the genetic code.

In 1959 Arthur Kornberg was a recipient of the Nobel prize for Physiology or Medicine based on his discovery of “the mechanisms in the biological synthesis of deoxyribonucleic acid” (DNA polymerase) together with Dr. Severo Ochoa of New York University. In the next 20 years Stanford University Department of Biochemistry became a top rated graduate program in biochemistry. Today, the Pfeffer Lab is distinguished for research into how human cells put receptors in the right place through Rab GTPases that regulate all aspects of receptor trafficking. Steve Elledge (1984-1989) at Harvard University is one of  its graduates from the 1980s.

Transcription –RNA and the ribosome

In 2006, Roger Kornberg was awarded the Nobel Prize in Chemistry for identifying the role of RNA polymerase II and other proteins in transcribing DNA. He says that the process is something akin to a machine. “It has moving parts which function in synchrony, in appropriate sequence and in synchrony with one another”. The Kornbergs were the tenth family with closely-related Nobel laureates.  The 2009 Nobel Prize in Chemistry was awarded to Venki Ramakrishnan, Tom Steitz, and Ada Yonath for crystallographic studies of the ribosome. The atomic resolution structures of the ribosomal subunits provide an extraordinary context for understanding one of the most fundamental aspects of cellular function: protein synthesis. Research on protein synthesis began with studies of microsomes, and three papers were published on the atomic resolution structures of the 50S and 30S the atomic resolution of structures of ribosomal subnits in 2000. Perhaps the most remarkable and inexplicable feature of ribosome structure is that two-thirds of the mass is composed of large RNA molecules, the 5S, 16S, and 23S ribosomal RNAs, and the remaining third is distributed among ~50 relatively small and innocuous proteins. The first step on the road to solving the ribosome structure was determining the primary structure of the 16S and 23S RNAs in Harry Noller’s laboratory. The sequences were rapidly followed by secondary structure models for the folding of the two ribosomal RNAs, in collaboration with Carl Woese, bringing the ribosome structure into two dimensions. The RNA secondary structures are characterized by an elaborate series of helices and loops of unknown structure, but other than the insights offered by the structure of transfer RNA (tRNA), there was no way to think about folding these structures into three dimensions. The first three-dimensional images of the ribosome emerged from Jim Lake’s reconstructions from electron microscopy (EM) (Lake, 1976).

Ada Yonath reported the first crystals of the 50S ribosomal subunit in 1980, a crucial step that would require almost 20 years to bring to fruition (Yonath et al., 1980). Yonath’s group introduced the innovative use of ribosomes from extremophilic organisms. Peter Moore and Don Engelman applied neutron scattering techniques to determine the relative positions of ribosomal proteins in the 30S ribosomal subunit at the same time. Elegant chemical footprinting studies from the Noller laboratory provided a basis for intertwining the RNA among the ribosomal proteins, but there was still insufficient information to produce a high resolution structure, but Venki Ramakrishnan, in Peter Moore’s laboratory did it with deuterated ribosome reconstitutions. Then the Yale group was ramping up its work on the H. marismortui crystals of the 50S subunit. Peter Moore had recruited long-time colleague Tom Steitz to work on this problem and Steitz was about to complete the final event in the pentathlon of Crick’s dogma, having solved critical structures of DNA polymerases, the glutaminyl tRNA-tRNA synthetase complex, HIV reverse transcriptase, and T7 RNA polymerase. In 1999 Steitz, Ramakrishnan, and Yonath all presented electron density maps of subunits at approximately 5 Å resolution, and the Noller group presented 10 Å electron density maps of the Thermus 70S ribosome. Peter Moore aptly paraphrased Churchill, telling attendees that this was not the end, but the end of the beginning. Almost every nucleotide in the RNA is involved in multiple stabilizing interactions that form the monolithic tertiary structure at the heart of the ribosome.
Williamson J. The ribosome at atomic resolution. Cell 2009; 139:1041-1043.    http://dx.doi.org/10.1016/j.cell.2009.11.028/      http://www.sciencedirect.com/science/article/pii/S0092867409014536

This opened the door to new therapies.  For example, in 2010 it was reported that Numerous human genes display dual coding within alternatively spliced regions, which give rise to distinct protein products that include segments translated in more than one reading frame. To resolve the ensuing protein structural puzzle, we identified human genes with alternative splice variants comprising a dual coding region at least 75 nucleotides in length and analyzed the structural status of the protein segments they encode. The inspection of their amino acid composition and predictions by the IUPred and PONDR® VSL2 algorithms suggest a high propensity for structural disorder in dual-coding regions.
Kovacs E, Tompa P, liliom K, and Kalmar L. Dual coding in alternative reading frames correlates with intrinsic protein disorder. PNAS 2010.   http://www.jstor.org/stable/25664997   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851785
http://www.pnas.org/content/107/12/5429.full.pdf


In 2012, it was shown that drug-bound ribosomes can synthesize a distinct subset of cellular polypeptides. The structure of a protein defines its ability to thread through the antibiotic-obstructed tunnel. Synthesis of certain polypeptides that initially bypass translational arrest can be stopped at later stages of elongation while translation of some proteins goes to completion. (Kannan K, Vasquez-Laslop N, and Mankin AS. Selective Protein Synthesis by Ribosomes with a Drug-Obstructed Exit Tunnel. Cell 2012; 151; 508-520.) http://dx.doi.org/10.1016/j.cell.2012.09.018     http://www.sciencedirect.com/science/article/pii/S0092867412011257

Mobility of genetic elements

Barbara McClintock received the Nobel Prize for Medicine for the discovery of the mobility of genetic elements, work that been done in that period. When transposons were demonstrated in bacteria, yeast and other organisms, Barbara rose to a stratospheric level in the general esteem of the scientific world, but she was uncomfortable about the honors. It was sufficient to have her work understood and acknowledged. Prof. Howard Green said of her, “There are scientists whose discoveries greatly transcend their personalities and their humanity. But those in the future who will know of Barbara only her discoveries will know only her shadow”.
“In Memoriam – Barbara McClintock”. Nobelprize.org. 5 Feb 2013   http://www.nobelprize.org/nobel_prizes/medicine/laureates/1983/mcclintock-article.html/

She introduced her Nobel Lecture in 1983 with the following observation: “An experiment conducted in the mid-nineteen forties prepared me to expect unusual responses of a genome to challenges for which the genome is unprepared to meet in an orderly, programmed manner. In most known instances of this kind, the types of response were not predictable in advance of initial observations of them. It was necessary to subject the genome repeatedly to the same challenge in order to observe and appreciate the nature of the changes it induces…a highly programmed sequence of events within the cell that serves to cushion the effects of the shock. Some sensing mechanism must be present in these instances to alert the cell to imminent danger, and to set in motion the orderly sequence of events that will mitigate this danger”. She goes on to consider “early studies that revealed programmed responses to threats that are initiated within the genome itself, as well as others similarly initiated, that lead to new and irreversible genomic modifications. These latter responses, now known to occur in many organisms, are significant for appreciating how a genome may reorganize itself when faced with a difficulty for which it is unprepared”.

An experiment with Zea conducted in the summer of 1944 alerted her to the mobility of specific components of genomes involved the entrance of a newly ruptured end of a chromosome into a telophase nucleus. This experiment commenced with the growing of approximately 450 plants in the summer of 1944, each of which had started its development with a zygote that had received from each parent a chromosome with a newly ruptured end of one of its arms. The design of the experiment required that each plant be self-pollinated to isolate from the self-pollinated progeny new mutants that were expected to appear, and confine them to locations within the ruptured arm of a chromosome. Each mutant was expected to reveal the phenotype produced by a minute homozygous deficiency. Their modes of origin could be projected from the known behavior of broken ends of chromosomes in successive mitoses. Forty kernels from each self-pollinated ear were sown in a seedling bench in the greenhouse during the winter of 1944-45.

Some seedling mutants of the type expected overshadowed by segregants exhibiting bizarre phenotypes. These were variegated for type and degree of expression of a gene. Those variegated expressions given by genes associated with chlorophyll development were startingly conspicuous. Within any one progeny chlorophyll intensities, and their pattern of distribution in the seedling leaves, were alike. Between progenies, however, both the type and the pattern differed widely.

The effect of X-rays on chromosomes

Initial studies of broken ends of chromosomes began in the summer of 1931. By 1931, means of studying the beads on a string hypothesis was provided by newly developed methods of examining the ten chromosomes of the maize complement in microsporocytes in meiosis. The ten bivalent chromosomes are elongated in comparison to their metaphase lengths. Each chromosome

  • is identifiable by its relative length,
  • by the location of its centromere, which is readily observed at the pachytene stage, and
  • by the individuality of the chromomeres strung along the length of each chromosome.

At that time maize provided the best material for locating known genes along a chromosome arm, and also for precisely determining the break points in chromosomes that had undergone various types of rearrangement, such as translocations, inversions, etc.
The recessive phenotypes in the examined plants arose from loss of a segment of a chromosome that carried the wild-type allele, and X-rays were responsible for inducing these deficiencies. A conclusion of basic significance could be drawn from these observations:

  1. broken ends of chromosomes will fuse, 2-by-2, and
  2. any broken end with any other broken end.

This principle has been amply proved in a series of experiments conducted over the years. In all such instances the break must sever both strands of the DNA double helix. This is a “double-strand break” in modern terminology. That two such broken ends entering a telophase nucleus will find each other and fuse, regardless of the initial distance that separates them, soon became apparent.

During the summer of 1931 she had seen plants in the maize field that showed variegation patterns resembling the one described for Nicotiana.  Dr. McClintock was interested in selecting the variegated plants to determine the presence of a ring chromosome in each, and in the summer of 1932 with Dr. Stadler’s generous cooperation from Missouri, she had the opportunity to examine such plants. Each plant had a ring chromosome, but It was the behavior of this ring that proved to be significant. It revealed several basic phenomena. The following was noted:

In the majority of mitoses

  • replication of the ring chromosome produced two chromatids completely free from each other
  • could separate without difficulty in the following anaphase.
  • sister strand exchanges do occur between replicated or replicating chromatids
  • the frequency of such events increases with increase in the size of the ring.
  • these exchanges produce a double-size ring with two centromeres.
  • Mechanical rupture occurs in each of the two chromatid bridges formed at anaphase by passage of the two centromeres on the double-size ring to opposite poles of the mitotic spindle.
  • The location of a break can be at any one position along any one bridge.
  • The broken ends entering a telophase nucleus then fuse.
  • The size and content of each newly constructed ring depend on the position of the rupture that had occurred in each bridge.
  1. The conclusion was that cells sense the presence in their nuclei of ruptured ends of chromosomes
  2. then activate a mechanism that will bring together and then unite these ends
  3. this will occur regardless of the initial distance in a telophase nucleus that separated the ruptured ends.

The ability of a cell to

  • sense these broken ends,
  • to direct them toward each other, and
  • then to unite them so that the union of the two DNA strands is correctly oriented,
  • is a particularly revealing example of the sensitivity of cells to all that is going on within them.

Evidence from gave unequivocal support for the conclusion that broken ends will find each other and fuse. The challenge is met by a programmed response. This may be necessary, as

  1. both accidental breaks and
  2. programmed breaks may be frequent.
  3. If not repaired, such breaks could lead to genomic deficiencies having serious consequences.

A cell capable of repairing a ruptured end of a chromosome must sense the presence of this end in its nucleus. This sensing

  • activates a mechanism that is required for replacing the ruptured end with a functional telomere.
  • that such a mechanism must exist was revealed by a mutant that arose in the stocks.
  • this mutant would not allow the repair mechanism to operate in the cells of the plant.

Entrance of a newly ruptured end of a chromosome into the zygote is followed by the chromatid type of breakage-fusion-bridge cycle throughout mitoses in the developing plant.
This suggested that the repair mechanism in the maize strains is repressed in cells producing

  • the male and female gametophytes and
  • also in the endosperm,
  • but is activated in the embryo.

The extent of trauma perceived by cells

  • whose nuclei receive a single newly ruptured end of a chromosome that the cell cannot repair,
  • and the speed with which this trauma is registered, was not appreciated until the winter of 1944-45.

By 1947 it was learned that the bizarre variegated phenotypes that segregated in many of the self-pollinated progenies grown on the seedling bench in the fall and winter of 1944-45, were due to the action of transposable elements. It seemed clear that

  • these elements must have been present in the genome,
  • and in a silent state previous to an event that activated one or another of them.

She concluded that some traumatic event was responsible for these activations. The unique event in the history of these plants relates to their origin. Both parents of the plants grown in 1944 had contributed a chromosome with a newly ruptured end to the zygote that gave rise to each of these plants.
Detection of silent elements is now made possible with the aid of DNA cloning method. Silent AC (Activator) elements, as well as modified derivatives of them, have already been detected in several strains of maize. When other transposable elements are cloned it will be possible to compare their structural and numerical differences among various strains of maize. In any one strain of maize the number of silent but potentially transposable elements, as well as other repetitious DNAs, may be observed to change, and most probably in response to challenges not yet recognized.
Telomeres are especially adapted to replicate free ends of chromosomes. When no telomere is present, attempts to replicate this uncapped end may be responsible for the apparent “fusions” of the replicated chromatids at the position of the previous break as well as for perpetuating the chromatid type of breakage-fusion-bridge cycle in successive mitoses.
In conclusion, a genome may react to conditions for which it is unprepared, but to which it responds in a totally unexpected manner. Among these is

  • the extraordinary response of the maize genome to entrance of a single ruptured end of a chromosome into a telophase nucleus.
  • It was this event that was responsible for activations of potentially transposable elements that are carried in a silent state in the maize genome.
  • The mobility of these activated elements allows them to enter different gene loci and to take over control of action of the gene wherever one may enter.

Because the broken end of a chromosome entering a telophase nucleus can initiate activations of a number of different potentially transposable elements,

  • the modifications these elements induce in the genome may be explored readily.

In addition to

modifying gene action, these elements can

  • restructure the genome at various levels,
  • from small changes involving a few nucleotides,
  • to gross modifications involving large segments of chromosomes, such as
  1. duplications,
  2. deficiencies,
  3. inversions,
  4. and other reorganizations.

In the future attention undoubtedly will be centered on the genome, and with greater appreciation of its significance as a highly sensitive organ of the cell,

  • monitoring genomic activities and correcting common errors,
  • sensing the unusual and unexpected events,
  • and responding to them,
  • often by restructuring the genome.

We know about the elements available for such restructuring. We know nothing, however, about

  • how the cell senses danger and instigates responses to it that often are truly remarkable.

Source: 1983 Nobel Lecture. Barbara McClintock. THE SIGNIFICANCE OF RESPONSES OF THE GENOME TO CHALLENGE.

In 2009 the Nobel Prize in Physiology or Medicine was awarded to Elizabeth Blackburn, Carol Greider and Jack Szoztak for the discovery of Telomerase. This recognition came less than a decade after the completion of the Human Genome Project previously discussed. Prof. Blackburn acknowledges a strong influence coming from the work of Barbara McClintock. The discovery is tied to the pond organism Tetrahymena thermophila, and studies of yeast cells. Blackburn was drawn to science after reading the biography of Marie Curie by her daughter, Irina, as a child. She recalls that her Master’s mentor while studying the metabolism of glutamine in the rat liver, thought that every experiment should have the beauty and simplicity of a Mozart sonata. She did her PhD at the distinguished Laboratory for Molecular Biology at Cambridge, the epicenter of molecular biology sequencing the regions of bacteriophage phiX 174, a single stranded DNA bacteriophage. Using Fred Sanger’s methods to piece together RNA sequences she showed the first sequence of a 48 nucleotide fragment to her mathematical-gifted Cambridge cousin, who pointed out repeats of DNA sequence patterns! She worked on the sequencing of the DNA at the terminal regions of  the short “minichromosomes” of the ciliated protozoan Tetrahymena thermophile at Yale in 1975. She continued her research begun at Yale at UCSF funded by the NIH based on an intriguing audiogram showing telomeric DNA in Tetrahymena.
I describe the work as follows:

  • Prof. Blackburn incorporated 32P isotope labelled deoxynucleoside residues into the rDNA molecules for DNA repair enzymatic reactions and found that
  • the end regions were selectively labeled by combinations of 32P isotope radiolabled nucleoside triphosphate, and by mid-year she had an audiogram of the depurination products.
  • The audiogram showed sequences of 4 cytosine residues flanked by either an adenosine or a guanosine residue.
  • In 1976 she had deduced a sequence consisting of a tandem array of CCCAA repeats, and subsequently separated the products on a denaturing gel electrophoresis that appeared as tiger stripes extending up the gel.
  • The size of each band was 6 bases more than the band below it.

Telomere must have a telomerase!

The discovery of the telomerase enzyme activity was done by the Prize co-awardee, Carol Greider. They were trying to decipher the structure right at the termini of telomeres of both cliliated protozoans and yeast plasmids. The view that in mammalian telomeres there is a long protruding G-rich strand does not take into account the clear evidence for the short C strand repeat oligonucleotides that she discovered. This was found for both the Tetrahymena rDNA minichromosome molecules and linear plasmids purified from yeast.
In contrast to nucleosomal regions of chromosomes, special regions of DNA, for example

  • promoters that must bind transcription initiation factors that control transcription, have proteins other than the histones on them.
  • The telomeric repeat tract turned out to be such a non-nucleosomal region.

They  found that by clipping up chromatin using an enzyme that cuts the linker between neighboring nucleosomes,

  • it cut up the bulk of the DNA into nucleosome-sized pieces
  • but left the telomeric DNA tract as a single protected chunk.

The resulting complex of the telomeric DNA tract plus its bound cargo of protective proteins behaved very differently, from nucleosomal chromatin, and concluded that it had no histones or nucleosomes.

Any evidence for a protein on the bulk of the rDNA molecule ends, such as their behavior in gel electrophoresis and the appearance of the rDNA molecules under the electron microscope, was conspicuously lacking. This was reassuring that there was no covalently attached protein at the very ends of this minichoromosome. Despite considerable work, she was unable to determine what protein(s) would co-purify with the telomeric repeat tract DNA of Tetrahymena. It was yeast genetics and approaches done by others that turned out to provide the next great leaps forward in understanding telomeric proteins. Carol Greider, her colleague, noticed the need to scale up the telomerase activity preparations and they used a very large glass column for preparative gel filtration chromatography.

Jack W Szostak at the Howard Hughes Medical Institue at Harvard shared in the 2009 Nobel Prize. He became interested in molecular biology taking a course on the frontiers of Molecular Biology and reading about the experiments of Meselson-Stahl barely a decade earlier, and learned how the genetic code had been unraveled. The fact that one could deduce, from measurements of the radioactivity in fractions from a centrifuge tube, the molecular details of DNA replication, transcription and translation was astonishing. A highlight of his time at McGill was the open-book, open-discussion final exam in this class, in which the questions required the intense collaboration of groups of students.

At Cornell, Ithaca, he collaborated with  John Stiles and they came up with a specific idea to chemically synthesize a DNA oligonucleotide of sufficient length that it would hybridize to a single sequence within the yeast genome, and then to use it as an mRNA and gene specific probe. At the time, there was only one short segment of the yeast genome for which the DNA sequence was known,

  • the region coding for the N-terminus of the iso-1 cytochrome c protein,

intensively studied by Fred Sherman
The Sherman lab, in a tour de force of genetics and protein chemistry, had isolated

  • double-frameshift mutants in which the N-terminal region of the protein was translated from out-of-frame codons.
  • Protein sequencing of the wild type and frame-shifted mutants allowed them to deduce 44 nucleotides of DNA sequence.

If they could prepare a synthetic oligonucleotide that was complementary to the coding sequence, they could use it to detect the cytochrome-c mRNA and gene. At the time, essentially all experiments on mRNA were done on total cellular mRNA. Ray Wu was already well known for determining the sequence of the sticky ends of phage lambda, the first ever DNA to be sequenced, and his lab was deeply involved in the study of enzymes that could be used to manipulate and sequence DNA more effectively, but would not take on a project from another laboratory. So John went to nearby Rochester to do postdoctoral work with Sherman, and he was able to transfer to Ray Wu’s laboratory. In order to carry out his work, Ray Wu sent him to Saran Narang’s lab in Ottawa, and he received training there under Keichi Itakura, who synthesized the Insulin gene. A few months later, he received several milligrams of our long sought 15-mer. In collaboration with John Stiles and Fred Sherman, who sent us RNA and DNA samples from appropriate yeast strains, they were able to use the labeled 15-mer as a probe to detect the cyc1 mRNA, and later the gene itself. He notes that one of the delights of the world of science is that it is filled with people of good will who are more than happy to assist a student or colleague by teaching a technique or discussing a problem. He remained in Ray’s lab after completion of the PhD upon the arrival of Rodney Rothstein from Sherman’s lab in Rochester, who introduced him to yeast genetics, and he was prepared for the next decade of work on yeast.

  • first in recombination studies, and
  • later in telomere studies and other aspects of yeast biology.

His studies of recombination in yeast were enabled by the discovery, in Gerry Fink’s lab at Cornell, of a way to introduce foreign DNA into yeast. These pioneering studies of yeast transformation showed that circular plasmid DNA molecules could on occasion become integrated into yeast chromosomal DNA by homologous recombination.

  • His studies of unequal sister chromatid exchange in rDNA locus resulted in his first publication in the field of recombination.

The idea that you could increase transformation frequency by cutting the input DNA was pleasingly counterintuitive and led us to continue our exploration of this phenomenon. He gained an appointment to the Sidney-Farber Cancer Institute due to the interest of Prof. Ruth Sager, who gathered together a great group of young investigators. In work spearheaded by his first graduate student, Terry Orr-Weaver, on

  • double-strand breaks in DNA
  • and their repair by recombination (and continuing interaction with Rod Rothstein),
  • they were attracted to what kinds of reactions occur at the DNA ends.

It was at a Gordon Conference that he was excited hearing a talk by Elizabeth Blackburn on her work on telomeres in Tetrahymena.

  • This led to a collaboration testing the ability of Tetrahymena telomers to function in yeast.
  • He performed the experiments himself, and experienced the thrill of being the first to know that our wild idea had worked.
  • It was clear from that point on that a door had been opened and that they were going to be able to learn a lot about telomere function from studies in yeast.
  • Within a short time he was able to clone bona fide yeast telomeres, and (in a continuation of the collaboration with Liz Blackburn’s lab)
  • they obtained the critical sequence information that led (them) to propose the existence of the key enzyme, telomerase.

A fanciful depiction evoking both telomere dynamics and telomere researchers, done by the artist Julie Newdoll in 2008, elicits the idea of a telomere as an ancient Sumarian temple-like hive, tended by a swarm of ancient Sumarian Bee-goddesses against a background of clay tablets inscribed with DNA sequencing gel-like bands.
Dr. Blackburn recalls owing much to Barbara McClintock for her scientific findings, but also, Barbara McClintock also gave her advice in a conversation with her in 1977, during which

  • she had unexpected findings with the rDNA end sequences.
  • Dr. McClintock urged her to trust in intuition about the scientific research results.

This advice was surprising then because intuitive thinking was not something that she accepted to be a valid aspect of being a biology researcher.
MLA style: “Elizabeth H. Blackburn – Biographical”. Nobelprize.org. 5 Feb 2013. http://www.nobelprize.org/nobel_prizes/medicine/laureates/2009/blackburn.html

Summary:

In this Part I of a series of 3, I have described the

  • emergence of Molecular Biology and
  • closely allied work on the mechanism of Cell Replication and
  • the dependence of metabolic processes on proteins and enzymatic conversions through a surge of
  • post WWII research that gave birth to centers for basic science research in biology and medicine in both US and in England, which was preceded by work in prewar Germany. This is to be followed by further developments related to the Human Genome Project.
  • Transcription initiation (Photo credit: Wikipedia)
  • Schematic relationship between biochemistry, genetics, and molecular biology (Photo credit: Wikipedia)
  • Central dogma of molecular biology (Photo credit: Wikipedia)

 

Transcription initiation

Transcription initiation (Photo credit: Wikipedia)

Schematic relationship between biochemistry, g...

Schematic relationship between biochemistry, genetics, and molecular biology (Photo credit: Wikipedia)

Central dogma of molecular biology

Central dogma of molecular biology (Photo credit: Wikipedia)

 

 

 

                        Nucleotides_1.svg

 

 

 

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Read Full Post »


Reporter: Ritu Saxena, Ph.D.

On December 4, 2012, molecular diagnostic firm Invivoscribe Technologies launched a personalized medicine company. Genection is offering both routine and esoteric genetic tests, exome and whole-genome sequencing, cancer somatic mutation testing, and pharmacogenomics.

Image

Because the Genection model is not payor-driven, it said, it can provide doctors access to genetic tests that are currently unavailable, overlooked, or inaccessible through their patients’ health plans and healthcare institutions.

The privately held company added that it has agreements in place with several CLIA- and CAP-certified laboratories, including ARUP Laboratories, Foundation Medicine, Cypher Genomics, Invivoscribe’s wholly owned subsidiary the Laboratory for Personalized Molecular Medicine and LPMM’s laboratory in Martinsried, Germany. It also has relationships with Illumina and Ambry Genetics and agreements with “a consortium” of genetic counselors.

“In order to make personalized molecular medicine a clinical reality, new platforms need to be developed for the delivery of healthcare. Genection’s mission seeks to accelerate this adoption process,” Genection Chief Medical Officer Bradley Patay said in a statement. “The combination of CLIA-validated genetic testing, whole-exome or whole-genome sequencing, and broad targeted assays, along with critical bioinformatics, analytic tools, and interpretative guidelines will contribute to timely definitive diagnoses for patients with rare, unexplained diseases or complex diseases; in essence, this integration will speed delivery of genomic test results and improve patient care.”

The company profile states that because the cost of genomic sequencing has declined steeply, utilizing deep sequencing of tumors, doctors can now offer targeted treatments to the specific type of cancer for each patient. This personalized approach may offer better treatment options that are tailored for each individual versus conventional approaches.  For example, The Cancer Genome Atlas Research Network found a potential therapeutic target in most squamous cell lung cancers. Genetic testing would also be able to provide insight on drug’s effectiveness and help a physician tailor the dosage and/or select another drug if it’s determined that you have a genetic variant that could affect the drug’s efficacy.

Source:

http://www.genomeweb.com//node/1159221?hq_e=el&hq_m=1425051&hq_l=3&hq_v=e618131fd2

Invivoscribe Technologies: http://www.invivoscribe.com/

Genection: http://www.genection.com/

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Sunitinib brings Adult Acute Lymphoblastic Leukemia (ALL) to Remission – RNA Sequencing – FLT3 Receptor Blockade

Curator: Aviva Lev-Ari, PhD, RN

Updated 11/13/2013

Pazopanib versus Sunitinib in Renal Cancer

N Engl J Med 2013; 369:1968-1970November 14, 2013DOI: 10.1056/NEJMc1311795

Article

To the Editor:

Cancer treatments are expensive. The estimation of the total cost can be challenging because of several factors such as efficacy, toxicity, and the costs and duration of supportive care and end-of-life care. Motzer et al. (Aug. 22 issue)1 report similar efficacy but a favorable safety and quality-of-life profile and less medical resource utilization with pazopanib as compared with sunitinib in first-line therapy for metastatic renal cancer. Since oncology is becoming an increasingly value-based specialty, we wanted to highlight another important aspect of this trial. Pazopanib appears to be favorable not only in terms of safety and quality of life, but also in terms of overall cost. A 30-day supply of pazopanib (at a dose of 800 mg daily) ranges from $3,500 to $8,556, whereas a 30-day supply of sunitinib (at a dose of 50 mg daily) ranges from $4,500 to $13,559.2 The total cost of pazopanib during the median progression-free survival of 8.4 months is $29,400 to $71,870, and the total cost of sunitinib during the median progression-free survival of 9.5 months is $42,750 to $127,454. Less toxicity and less medical resource utilization with pazopanib will most likely further lower the overall costs of treatment with this agent. Comparative-effectiveness trials hold great promise for maximizing patient safety, improving treatment outcomes, and reducing costs.

Ryan Ramaekers, M.D.
Mark Tharnish, Pharm.D.
M. Sitki Copur, M.D.
Saint Francis Cancer Treatment Center, Grand Island, NE
mcopur@sfmc-gi.org

No potential conflict of interest relevant to this letter was reported.

2 References

To the Editor:

Motzer et al. report a combined analysis of two open-label noninferiority trials (927 patients in the original trial and 183 patients in a second trial), each of which compared pazopanib with sunitinib with respect to progression-free survival in renal-cell carcinoma. Quality-of-life outcomes were subjective.

Analysis of noninferiority trials is notoriously difficult.1,2 The authors’ analysis of the trials, which was open-label because of the different administration schedules of the drugs, presents problems in interpreting progression-free survival and quality of life. The studies define disease progression differently. The larger study defined progression-free survival according to independent review. The protocol for the smaller study states that progression-free survival “will be summarized . . . based on the investigator assessment.” Inference from subjective outcomes in unmasked trials (e.g., quality of life in both studies and progression-free survival in the smaller study and therefore in the combined analysis) is subject to well-known bias. Moreover, the article does not state how many of the 379 participants (34%) who discontinued the intervention before death or disease progression (see Fig. S2 in the Supplementary Appendix, available with the full text of the article at NEJM.org) were assessed for progression-free survival. A fair comparison must use rigorous methods to handle missing data.3 Since the article did not deal appropriately with missing data, its conclusions regarding noninferiority are uninterpretable.

Janet Wittes, Ph.D.
Statistics Collaborative, Washington, DC
janet@statcollab.com

Dr. Wittes reports that her company, Statistics Collaborative, has consulting agreements with both GlaxoSmithKline and Pfizer, the manufacturers of the drugs discussed in the article by Motzer et al. In addition, Statistics Collaborative has contracts with several other companies that produce drugs for patients with cancer. No other potential conflict of interest relevant to this letter was reported.

3 References

To the Editor:

Motzer et al. state that “the results of the progression-free survival analysis in the per-protocol population were consistent with the results of the primary analysis.” However, the predefined margin of noninferiority (<1.25) was not met. The upper limit of the confidence interval (1.255) was clearly above the defined threshold.1 In a noninferiority trial, the use of the intention-to-treat population is generally nonconservative,2 the full analysis set and the per-protocol analysis set are considered to have equal importance, and the use of the intention-to-treat population should lead to similar conclusions for a robust interpretation.3 Thus, it is surprising that the authors did not come to or discuss the same conclusions as that of the French National Authority for Health4: “serious doubt exists about the noninferiority result of pazopanib compared to sunitinib” and “the clinical significance of the noninferiority threshold defined in the protocol was an efficacy loss of 2.2 months in the median progression-free survival. This is too large for patients.”

Jochen Casper, M.D.
Silke Schumann-Binarsch, M.D.
Claus-Henning Köhne, M.D.
Klinikum Oldenburg, Oldenburg, Germany
casper.jochen@klinikum-oldenburg.de

Dr. Casper reports receiving consulting fees from Bayer, Novartis, and Pfizer and speaking fees from Novartis and Pfizer. No other potential conflict of interest relevant to this letter was reported.

4 References

The authors reply: In reply to Ramaekers et al.: we agree that decisions regarding the provision of health care include economic evaluations to identify treatments that provide the best clinical benefit at an acceptable cost.

To clarify a point in the letter by Wittes: the primary end point of this phase 3 trial was progression-free survival evaluated by an independent review committee; these data were assessed for all 1110 patients from both trials. This is specified in the protocol. The consistency of the quality-of-life results with the observed differences in the safety profiles for the two drugs speaks to the absence of bias in the quality-of-life outcome. The number of patients in whom follow-up ended before progression was assessed by the independent review committee was balanced between the two groups: 156 patients in the pazopanib group (28%) and 168 patients in the sunitinib group (30%). To Wittes’s final point regarding rigorous methods to handle missing data: the algorithm for assigning disease-progression and censoring dates followed the Guidance for Industry of the Food and Drug Administration1 and is included in the protocol of our article.

In reply to Casper et al.: there is no consensus regarding whether the per-protocol population is more conservative than the intention-to-treat population for the noninferiority analysis.2,3Reviews of noninferiority trials indicate that the per-protocol population is not generally more conservative than the intention-to-treat population, and there are scenarios in which the per-protocol analysis itself could introduce bias.3 A systematic review indicated that more than 70% of published findings from noninferiority trials in oncology show results in only the intention-to-treat population and not in the per-protocol population.4 Our phase 3 trial had a single primary analysis in the intention-to-treat population, with the per-protocol population included as a key sensitivity analysis, as supported by Fleming et al.5 No formal hypothesis testing was planned for the per-protocol population, nor was the trial powered for this. Consistency of the point estimates was desired to show an absence of bias due to the analysis population. This absence of bias was shown by the consistency of the hazard ratios (1.07 in the per-protocol analysis vs. 1.05 in the primary analysis). For an underpowered per-protocol comparison, it is inappropriate for Casper et al. to interpret that the upper bound that barely exceeded 1.25 in our per-protocol analysis is an indication of inconsistency of results across the two populations. The noninferiority margin was selected in consultation with oncology experts, and justification of the margin is in the protocol.

Robert J. Motzer, M.D.
Memorial Sloan-Kettering Cancer Center, New York, NY
motzerr@mskcc.org

Lauren McCann, Ph.D.
Keith Deen, M.S.
GlaxoSmithKline, Collegeville, PA

Since publication of their article, the authors report no further potential conflict of interest.

REFERENCES

Food and Drug Administration. Guidance for industry: clinical trial endpoints for the approval of cancer drugs and biologics. May 2007 (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071590.pdf).
Jones B, Jarvis P, Lewis JA, Ebbutt AF. Trials to assess equivalence: the importance of rigorous methods. BMJ 1996;313:36-39[Erratum, BMJ 1996;313:550.]
CrossRef | Web of Science | Medline
Brittain E, Lin D. A comparison of intent-to-treat and per-protocol results in antibiotic non-inferiority trials. Stat Med 2005;24:1-10
CrossRef | Web of Science | Medline
Tanaka S, Kinjo Y, Kataoka Y, Yoshimura K, Termukai S. Statistical issues and recommendations for noninferiority trials in oncology: a systematic review. Clin Cancer Res 2012;18:1837-1847
CrossRef | Web of Science | Medline
Fleming TR, Odem-Davis K, Rothmann MD, Li Shen Y. Some essential considerations in the design and conduct of non-inferiority trials. Clin Trials2011;8:432-439
CrossRef | Web of Science | Medline
SOURCE

Original Article Published on 7/9/2012

July 6, 2012 NY Times reports on a new approach based on DNA and RNA sequencing and a cancer drug for kidney cancer to bring REMISSION to Adult acute lymphoblastic leukemia (ALL).

On the lower left corner of this page – Watch the VIDEO

second-chance.html

Dr. Lukas Wartman, is a Cancer Researcher specializing in Leukemia. He suspected he had Leukemia, the very disease he had devoted his medical career to studying.

After years of treatment and two relapses of ALL, he has exhaused all conventional approaches to his disease. At Washington University in St. Louis, his colleagues in the lab, decoded Dr. Wartman’s genetic information by genome sequencing techniques t determine the genetic cause of his ALL. The team found an overactive gne, FLT3 on Chromosome 13. The gene was treated with pfizer’s Suntinib drug for advanced kidney cancer.

Blood samples free of ALL found in days after using the drug. As results were very promising, Pfizer, the drug’s maker who has turned down Dr. Wartman’s request for the drug under their compassionate use program, though he explained that his entire salary was only enough to pay for 7 1/2 months of Sutent (Suntinib). While he does not know why Pfizer gave him the drug finally, he suspects it was the plea of his Nurse Practitioner, Stephanie Bauer, NP.

Identification of the genetic cause for his ALL, thus discovering a breakthough in understanding and treatment for ALL in other patients, involved the following steps:

SAMPLE

two tissue samples taken from Dr. Wartman’s Bone marrow and skin cells

SEQUENCE

Extracts of DNA and RNA from Dr. Wartman’s cells, two types of genetic material tested

COMPARISON

DNA sequesnces showed genetic mutations possibly related to his ALL, none seemed treatable. However, RNA sequencing revealed that a normal Gene, FLT3, on cheomozome 13, was overactive in his leukemia cells

TARGETING

The FLT3 gene helps create new white blod cells in the bone marrow. Dr. Wartman’s marrow bone cells were covered with an extreme number of FLT3 receptors which possibly caused the growth of his leukemia.

TREATMENT – Receptor Blockade 

Drug known to block FLT3 receptor, Sunitinib, used for kedney cancer treatment, was given to Dr. Wartman. Two weeks after Dr, Wartman began taking the drug, tests revealed that his leukenia was in remission.

NEW MARKETS FOR FLT3  GENE BLOCKADE DRUG  – KIDNEY CANCER AND LEUKEMIA

Pfizer has NOW a NEW market for Sunitinib — All CANCER PATIENTS DIAGNOSED WITH Adult acute lymphoblastic leukemia (ALL) where an overactive FLT3 gene on chomosome 13 is found.

NEW TREATMENT OPTIONS FOR Adult acute lymphoblastic leukemia (ALL)

Thus, any (ALL) diagnosed patient needs to be tested for Chromosome 13, ONLY rather then the entire genome sequencing of the Patient. If FLT3 is not found overactive, THEN proceed with entire genome sequencing of the Patient. IF another gene is overactive FIND DRUG FOR RECEPTOR BLOCKADE.

SIZING THE MARKET FOR FLT3 BLOCKADE DRUGS: KIDNEY CANCER vs LEUKEMIA

The Market for Adult ALL is much bigger than the market for kidney cancer. Thus, this discovery regarding the remission of Dr. Wartman’s remission following two relapses is so significant for Pfizer and for any patient with the diagnosis of Adult ALL.

I recommend the reader to click on the links and follow the reactions of the public to this article in The New York Times.

http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?pagewanted=all

Read HUNDREDS of Comments by Cancer Patients and the readers of The New York Times Health Section

http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?pagewanted=all#commentsContainer

 

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Author: Ritu Saxena, PhD

Updated on July 5, 2013 (research article published in New England Journal of Medicine regarding the role of SALL4 gene in aggressive hepatocellular carcinoma)

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The incidence of HCC varies considerably with the geographic area because of differences in the major causative factors. Chronic hepatitis B and C, mostly in the cirrhotic stage, are responsible for the great majority of cases of HCC worldwide.

Hepatitis B and C viruses (HBV/HCV) can be implicated in the development of HCC in an indirect way, through induction of chronic inflammation, or directly by means of viral proteins or, in the case of HBV, by creation of mutations by integration into the genome of the hepatocyte.http://www.wjso.com/content/3/1/27

With the advent of genome sequencing methodologies, it was about time that the scientists look clues within the genome of HCC tumor cells that would provide clues for disease progression via virus integration into the liver cells.

Two studies published in the recent issue of Nature Genetics (May 2012) explored the genome of HCC cells for genetic mutations that might be related to HBV and HCV highlighting the types of genetic mutations that underlie the liver cancer hepatocellular carcinoma, including forms of the disease related to hepatitis B and hepatitis C virus infection.

In the first study, Sung et al performed an extensive whole genome analysis using a large sample size of 88 Chinese individuals with HCC http://www.ncbi.nlm.nih.gov/pubmed?term=Genome-wide%20survey%20of%20recurrent%20HBV This was in the fact first unbiased, genome-wide, HBV-integration map in HCC leading to new recurrent integration sites and molecular mechanisms.

Although integration of viral DNA sequence within HCC genome has been reported in several studies, however, fewer cases of recurring mutations within genes during these integrations have been studied. The reason might be limited sample size in these studies. Tumor and non-tumor adjacent liver cells were surveyed in 81 HBV positive and 7 HBV negative HCC tumor samples. After the survey of whole genome of the 88 patients, several viral integration sites were discovered referred to as breakpoints. The breakpoints were found to be much more common in tumor than normal samples. Although the observed breakpoints were randomly distributed across the genome, a handful or frequently occurring sites referred to as ‘hotspots’ were discovered. The frequency of integration revealed that there were five genes with recurring integrations in HBV tumors- TERT, MLL4, CCNE5, SENP1, and ROCK1.

Apart from genome analysis, expression levels of the 5 genes implicated in the study were determined. In other words, the levels of proteins formed from the genes were compared and it was observed that samples with HBV integration had significantly higher level of protein expression of TERT, MLL4 and CCNE5 than the samples harboring no HBV integration sites. Although not statistically significant, overexpression of SENP1 and ROCK1 genes was also observed in HBV integration samples. This lead to an important conclusion from the study that the five genes that harbor recurrent HBV integrations might be implicated in HCC tumor development and that overexpression of these proteins is a probable molecular mechanism of tumorigenesis.

Interestingly, analysis of the HBV analysis revealed that almost 40% of the HBV genomes were cleaved at approximately 1,800 bp and then integrated into the human genome. The cleaved HBV sites had the necessary machinery (enhancers and ORF replication sites) for protein formation.

The study also confirmed the popular belief that HBV integrations might worsen the prognosis of HCC patients revealing a significant correlation between the number of HBV integrations and the survival of patients.An interesting observation from the study that had not been reported before was that HBV integration was associated with the occurrence of HCC at a younger age.

The study presented convincing evidence that chromosomal instability of HCC genome may originate from HBV integration.

A parallel study published in the same issue of Nature Genetics explored the genome of HCC tumors to gain insights into HBV and HCV-related genomic alterations. The research team sequenced whole-exon (protein forming genomic regions) of 27 liver tumors from 25 patients and compared with the corresponding genome sequences from matched white blood cell samples.

The study involved both HBV-related and HCV-related tumors along with two samples of tumors from individuals without HBV or HCV infection. The genome wide sequencing of HCC tumor cells revealed several mutations that included deletions and mutations of genes with predicted functional consequences. “Considering the high complexity and heterogeneity of [hepatocellular carcinomas] of both etiological and genetic aspects,” they concluded, “further molecular classification is required for appropriate diagnosis and therapy in personalized medicine.” Additionally, recurrent alterations were observed in the four genes – ARID1ARPS6KA3NFE2L2 and IRF2 that had not been previously described in HCC. The comprehensive mutation pattern observed in the study might be indicative of specific mutagenesis mechanisms occurring in tumor cells.

Authors said “Although no common somatic mutations were identified in the multicentric tumor pairs,” further stating “their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns.”The researchers suggested a major role of chromatin remodeling complexes and involvement of both interferon and oxidative stress pathways in hepatocellular malignant proliferation and transformation based on the genes showing recurrent mutations in the observed genes.

http://www.genomeweb.com/sequencing/studies-explore-genetics-behind-hepatitis-b-and-c-virus-associated-liver-cancers

http://www.ncbi.nlm.nih.gov/pubmed?term=Genome-wide%20survey%20of%20recurrent%20HBV

Thus, in both the studies new genes recurrently altered in HCC were identified along with uncovering some important clues relating to the molecular mechanism of virus-associated HCC.

Role of SALL4 in HCC

The oncofetal gene SALL4 is a marker of a subtype of HCC with progenitor-like features and is associated with a poor prognosis. Investigators at Cancer Science Institute of Singapore, National University of Singapore studied the role of oncofetal gene, SALL4 in HCC and the results were published were in a recent issue of New England Journal of Medicine ((Yong KJ, et al, Oncofetal Gene SALL4 in Aggressive Hepatocellular Carcinoma. http://www.ncbi.nlm.nih.gov/pubmed/23758232). Yong and colleagues (2013) screened specimens from patients with primary HCC for the expression of SALL4 and carried out a clinicopathological analysis. Loss-of-function studies were then performed to evaluate the role of SALL4 in hepatocarcinogenesis and its potential as a molecular target for therapy. Furthermore, in vitro functional and in vivo xenograft assays were performed to assess the therapeutic effects of a peptide that targets SALL4.

According to the results, SALL4 is an oncofetal protein that is expressed in the human fetal liver and silenced in the adult liver, but it is reexpressed in a subgroup of patients who have HCC and an unfavorable prognosis. Gene-expression analysis showed the enrichment of progenitor-like gene signatures with overexpression of proliferative and metastatic genes in SALL4-positive HCC. Loss-of-function studies confirmed the critical role of SALL4 in cell survival and tumorigenicity. The peptide targeting SALL4 blocked ­SALL4-corepressor interactions that released suppression of PTEN and inhibited tumor formation in xenograft assays in vivo. In conclusion, the results from the study indicate that SALL4 is a marker for a progenitor subclass of HCC with an aggressive phenotype. The absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in HCC.

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