Sat 810 Brothman - Association of Genetic Technologists

6/6/2015
2015 Gordon W. Dewald Lecture:
Cytogenomics, from yesterday through tomorrow
Arthur R. Brothman, Ph.D., FACMG
Professor of Pathology, Director of Cytogenomics, U of A, Tucson
Adjunct Professor of Pediatrics, Human Genetics and Pathology, U of U, Salt Lake City
Gordon Dewald 1943‐2010 Pioneer, innovator, colleague, and friend
Today’s talk:
Historical
Educational
Philosophical
Whimsical….
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People make the science (and the science often makes the person)
Objectives
• Discuss some of the changes we’ve seen in the field over the last four (!) decades
• Chromosomes – are conventional cytogenetic studies and FISH still necessary, prudent and sufficient? • CMA and NGS – where do these fit in in our diagnostic world?
• A few case examples, including an update on trends in prostate cancer research
(Very) quick history of cytogenetics
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Boveri‐Sutton chromosome theory ‐ 1904
Hsu – discovers hypotonic ‐ 1952
Tjio and Levan – “human 2n=46” ‐ 1956
Q‐banding (Caspersson) ‐ 1970
G‐banding (Seabright) ‐ 1971
FISH (Pinkel) ‐ 1986)
Sanger sequencing ‐ 1977
CGH (Kallioniemi ‐ 1993) CMA (Pinkel ‐ 1998)
Mayer and Farinelli ‐ “NGS” ‐ 2000
Human Genome Project complete ‐ 2001
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Ed’s project will be bring a piece of an asteroid back to earth
Ed and Art, while rafting the Grand Canyon in 2013
In college…
• A good friend, Ed Beshore, who is now senior staff scientist at the U of A and deputy P.I. on the OSIRIS REX mission, and I used to talk about our work – me looking through microscopes and him looking through telescopes…..
• He recently sent me “The Power of 10” (http://micro.magnet.fsu.edu/primer/java/scienceop
ticsu/powersof10/), which I used for many of the following images
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Cytogenomics, two components:
• Utilization for constitutional (germline) studies – CMA becoming accepted as first tier, already has replaced some studies
• Utilization for acquired (oncology) studies –
CMA gaining acceptance to complement conventional cytogenetics and FISH
This is “it” for techniques:
CMA (microarray)
NGS
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Replication “banding” by autoradiography, showing trisomy 13
From O.J. Miller
Clinical case – the “old technique” using spectral karyotyping
Morelli SH, Deubler DA, Brothman LJ, Carey JC, Brothman AR. Partial trisomy 17p detected by spectral karyotyping Clin Genet 1999: 55: 372–375. Detection of unbalanced rearrangement
Morelli SH, Deubler DA, Brothman LJ, Carey JC, Brothman AR. Partial
trisomy 17p detected by spectral karyotyping Clin Genet 1999: 55: 372–
375.
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Early on CMA was making cutting edge advances in constitutional genetics
FISH confirmation
control
patient
CMA in constitutional genetics: Rapid discovery of new
syndromes (almost every week!)
17q21 locus associated with autism and schizophrenia Moreno-del Luca et al AJHG 87:618, 2010
The value of
collaboration
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17q21 locus abnormalities showing high risk associations with autism
and schizophrenia. Moreno-del Luca et al AJHG 87:618, 2010
from: Lee, Iafrate, Brothman Nat Genet:39:S48,2007
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Beyond cytogenomics –
DNA sequencing
• First human genome sequence took ~11 years (1990 to 2001) to complete at a cost of ~$3‐billion.
• Now multiple genomes can be sequenced in a few days at a cost of <$2000.
• Initial technology: Sanger (capillary electrophoresis) sequencing (1975)
• Next generation (massive parallel sequencing) – 2008.
• Has led to commercially available testing, including prenatal, cell‐free screening for aneusomies.
Non‐invasive prenatal testing (NIPS)
• Cell free fetal DNA – Short fragments of DNA circulating in maternal serum, small proportion (<10%) of total DNA
– Detectable in maternal serum by 5th week; short half life (disappears within minutes postpartum)
• Companies currently offering NIPT (=NIPS) (massively parallel sequencing)
– Sequenom MaternaT21TM Plus
– Verinata Health ‐ verifi® Prenatal Test
– Natera PanoramaTM Prenatal Test
• The National Society of Genetic Counselors (NSGC) recommends genetic counseling with NIPS, and follow up of abnormal results with a conventional diagnostic procedure (amniocentesis)
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Recent position on “incidental (now called secondary) findings” (mutation
based, by whole genome sequencing) by ACMG
1. Pretesting counseling to define incidental findings
2. Limit initial clinical interpretations to well defined genes (56)
expect ~2% of the population to have a mutation in one
3. Patients and their families cannot “opt out” of knowing result
(“duty to warn” more important that autonomy); inform
parents and children of result. Negative result not “normal”
4. Genetic counseling for patients and their families critical
5. Updated in January, 2015”
– “Incidental findings” renamed “Secondary findings”
–Patients should have the choice to opt out of this secondary analysis.
–This patient choice should be “all or nothing;” the patient should not be allowed
to pick and choose which secondary genes should be analyzed.
–These guidelines will be frequently reviewed and updated – EVOLVING
Green et al. Genet Med 15(7):565, 2013 and Scheuner et al Genet Med 17(1):27, 2015
Cancer is a clonal disease and all cancers
have some genetic component
The first clear understanding of a mechanism
in cancer came from the identification of the
“Philadelphia chromosome” – t(9;22).
This resulted in the development of the first
“tailor made” drug to treat CML: Gleevac
(Imatinib) – a specific tyrosine kinase
inhibitor asssociated with the translocation
breakpoints – or fusion gene.
Cytogenetics, FISH and now CMA play major
roles in cancer diagnosis, prognosis and (as
noted above) treatment.
The Power of CMA in cancer detection
and prognostication
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But still – the complex abnormal karyotype still tells more than any molecular technique in many cases – atypical MDS progressed to AML with multiple abnormalities
A few of the many examples where we learn new information by FISH
MM: Gain of 1q Loss of 1q Loss of IGH
MDS: Loss of 5q, Gain of 5p = i(5p)
AML: inv(16)
FL: Complex t(14;18)
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My previous talks to AGT:
“Molecular Cytogenetic Models for Human Prostate Cancer” 22nd Annual American Genetic Technology Meeting, Portland, OR (June 1997).
“Chromopower: Where we stand on the learning curve”, 29th
Annual meeting of the Association of Genetic Technologists, Anaheim, CA, (June 2004).
The Prostate Exam
“Mr. Smith, this won’t hurt a bit”
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American Cancer Society statistics (US): comparison of prostate cancer data, 1997 vs. 2015
Number of new cases
(All sites)
Number of deaths (All Sites)
1997
334,500
(1,382,400) 41,800 (560,000)
2015
220,800
(1,658,370)
27,540 (580,430)
Incidence: for all cancers, increased 20%; prostate cancers have decreased 51% (ascertainment?)
Deaths: for all cancers, decreased 35%; prostate cancer deaths virtually unchanged (12.49% vs. 12.47%)
UCAP 27: 46, XY, del(10)(q25) Cytogenetics
GMA
By microarray:
del (10) (q11.1q21.1)
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Summary of gene fusions (ETS and RAF genes) in prostate cancer
Gu and Brothman, Ca Genet 204:47, 2011
Guangyu Gu
Normal Male Control (Peripheral Blood)
Prostate Cancer Touch Prep
146C
TMPRSS2/ERG
TMPRSS2/ERG fusion with interstitial deletion
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Exciting results indicating specific genes involved in prostate cancer progression, and also in metastases
Sequenced whole genomes of 7 prostate tumors/normal pairs
Genomic location, copy #
and rearrangements
Prostate tumor/normal
pairs confirmed several
genomic rearrangements
M F. Berger et al. Nature 470, 214-220 (2011)
Recurrent genomic copy number alterations in prostate cancer
Williams, Greer, Squire, Cancer Genetics 207, 373, 2014
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WGS – Mono and polyclonal seeding toward prostate metastasis (Gundem et al Nature April 2015) 10 prostate tumors sequenced, found either monoclonal seeding of new metastases or transfer of multiple tumor clones between metastatic sites
Mutations in tumor suppressor genes generally single events, mutations in AR involve multiple events
Case details, courtesy, Ryan Sprissler
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Case: 14 year old with chronic hypocalcemia with tetany since infancy Current treatment:
Calcitriol (1,25(OH)2 vitamin D3) 4mcg p.o. two times daily
Calcium citrate 3,800mg p.o. three times daily.
Cholecalciferol 3,000U p.o. daily
Calcium gluconate 7g, i.v. over 4 hours daily at home to control symptoms.
All insufficient to control blood calcium concentration
Very high levels of calcium excreted in feces
No calcium wasting in urine
Low 25(OH) vitamin D3 (5ng/mL) despite treatment.
Performed Whole Exome Sequencing on trio (parents and proband) Powerful analysis pipeline critical for sifting through data
Powerful analysis pipeline critical for sifting through data
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Summary
• LOTS of changes we’ve seen in the field over the last four decades
• Chromosomes – are conventional cytogenetic studies and FISH still necessary, prudent and sufficient? Absolutely – but we must evolve
• CMA and NGS – Here to stay and super powerful
• It’s the people (like YOU) who make the science!!!!!
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HAVE to give credit to Lisa Brothman!
Additional acknowledgements
• My previous and current cytogenetic labs – my extended family: EVMS (Norfolk), U of Utah (now ARUP, Salt Lake City) and U of Arizona (now Banner University Medicine, Tucson)
• Previous students, post‐docs, fellows, colleagues and technologists – too numerous to name but quick picture show:
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Warren G. Sanger, Ph.D.
October 6, 1945 ‐ February 5, 2015
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Xaiolin Zhu October 17th, 1954 ‐ April 8th, 2015 For Gordy – let’s all continue following his lead!
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