Download Moving Towards the Next Generation of Genetic Testing

DECEMBER 2015
ISSUE 139
IN THIS ISSUE
Moving Towards the
Next Generation of
Genetic Testing
News from HRLMP
Administration
Education News
News from Genetics
News from
Hematology
NEW – EMA test for
Diagnosis of HS
News from Pathology
Quality News
Research News –
Coming soon
Editorial Board:
Chemistry: Dr C Balion
Microbiology: Dr M Smieja
Pathology: Dr C Ross
Genetics: Dr E. McCready
Hematology: K Moffat
Editorial Office:
Co-Editors: Dr C Ross;
K Moffat
Snapshot of this edition:
•
•
Moving Towards the Next Generation of Genetic Testing
NEW - Eosin-5-Maleimide (EMA) Test for Diagnosis of Hereditary
Spherocytosis
HAPPY HOLIDAYS !!
Moving Towards the Next Generation of Genetic Testing
Since publication of the draft and completed human genome sequence in
2001 and 2004, respectively (Venter et al. 2001. Science, 291:1304-51;
International Human Genome Sequencing Consortium. 2001.Nature,
409:860-921; International Human Genome Sequencing Consortium.
2004. Nature, 431:931-45), the field of genetics and genomics has
experienced unprecedented growth that has led to an enhanced
understanding of how both heritable and acquired genetic mutations
contribute to population health. Expanding knowledge about the
molecular determinants of disease has further increased the pressure on
clinical genetics laboratories to provide more comprehensive testing of
an increasingly broad range of genetic variants.
The increasing utility of genetic testing coupled with both decreasing
costs and improved through-put of next generation sequencing (NGS)
technologies is driving forward a paradigm shift in laboratory genetics
practices. This shift is most apparent in an increasing trend away from
single gene analysis to the development of tests that enable analysis of
multiple genes simultaneously. Testing of single genes can have a high
diagnostic yield for disorders with specific syndromic features and/or
limited “causative” mutations. For example, the beta-globin gene (HBB)
is the only gene known to cause beta-thalassemia. Analysis of the HBB
gene for point mutations and deletions enables the accurate molecular
diagnosis of beta-thalassemia in over 99% of affected individuals.
However, there is limited utility of these single analyte tests for the
investigation of heterogeneous phenotypes that can be due to mutations
of any one of multiple genes. Noonan syndrome, for example, is known
THE EDITORIAL OFFICE WELCOMES YOUR FEEDBACK, SUGGESTIONS AND NEW IDEAS
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Lab Connections
to be caused by mutations of at least nine
different genes involved in the RAS-MAPK
pathway (PTPN11, SOS1, RAF1, KRAS, NRAS,
SHOC2, RIT1, BRAF and CBL); PTPN11 mutation
is the most common cause of Noonan
syndrome but genetic testing of this gene alone
only allows confirmation of the diagnosis in
50% of cases. Sequential analysis of associated
genes within the differential diagnosis list can
be prohibitively costly, require numerous blood
draws and evoke considerable anxiety for
patients who require repeated testing along
their "diagnostic odyssey". Development of
next generation sequencing (NGS) based assays
allow testing of all nine Noonan syndromeassociated genes in a single assay. This not only
improves the diagnostic yield of the test (up to
~82% of Noonan syndrome-associated alleles),
but also enables detection of mutations causing
other syndromes related to the RAS-MAPK
pathway including cardio-facio-cutaneous,
Noonan syndrome with multiple lentigines
(formerly LEOPARD) and Costello syndromes
(reviewed in Rauen. 2013. Annu Rev Genomics
Hum Genet, 14:355-69;Tidyman and
Rauen.2008. Expert Rev Mol Med, 10:e37).
stochastic effects. Sensitivity and specificity of
the assay can also be limited by the
bioinformatics tools used to analyze and
interpret the data. For example, an assay that
enables sequencing of all the known proteincoding genes (exome) can return 200-400,000
variants, most of which are benign and unlikely
to contribute to clinical features. Sequencing
all of the known protein-coding genes provides
a high degree of certainty that a “pathogenic”
mutation will be sequenced, if present. As
such, whole exome studies represent a
powerful investigational tool, particularly when
other genetic testing has been exhausted or
when a specific genetic diagnosis is not
apparent by clinical examination. Bioinformatic
tools are used to “filter” these variants and
enable a more focused analysis of variants most
likely to be pathogenic . While this makes
analysis of the data more streamlined and
manageable, it can also result in loss of relevant
data from the analysis pipeline. Therefore,
while exome tests relying on these types of
bioinformatic filters are extremely efficient as
screening tools, their utility in ruling-out a
genetic diagnosis is limited.
While next generation sequencing (NGS)
provides a unique opportunity to develop
genetic tests for large groups of genes, it is
important to note that not all NGS based tests
are created equal. The two most notable
factors that can influence test sensitivity and
specificity include the genomic regions targeted
by the test (sequence targets) and the number
of times a sequence is sampled (depth of
sequence coverage). Increasing the amount of
genomic sequence targeted by the test will
enhance the ability to detect a broader range of
mutations. However, this comes at the cost of
the number of times that any specific region is
sampled. Low depth of sequence coverage
increases the chance of false positive results
due to sequencing artifacts and of false
negative results or genotyping errors due to
Within the HRLMP there have been a number
of concerted efforts to incorporate NGS
technologies into the existing workflow.
Completed and ongoing test development
projects are anticipated to have significant
impact on the laboratory service by either
introducing new tests to the menu or by
improving efficiency and/or utility of existing
tests. In January of this year, BRCA1- and
BRCA2-associated hereditary breast and
ovarian cancer became the first test that was
transitioned to the new technology and has
served to “pave the trail” for the development
of other tests. Prior to the introduction of a
NGS protocol, BRCA1 and BRCA2 were
assessed by a more traditional approach that
required amplification of the entire BRCA1 and
BRCA2 protein-coding regions through 61
December 2015  Issue 139
independent reactions. This test was very
labour intensive and required two technologists
an average of two weeks to process 16
samples. Conversion of the test to NGS has
streamlined the testing considerably and
enables processing 24 samples with 1.5
technologists in the same period of time. The
entire coding region is sequenced to a very high
depth of coverage allowing for sensitivity and
specificity rates approaching 100%. The high
depth of coverage further enables detection of
partial or whole gene deletions/duplications
that may be missed using traditional
sequencing protocols, thus eliminating the
need for copy number evaluation using
multiplex ligation-dependent amplification
(MLPA).
Building on the success of the BRCA1 and
BRCA2 NGS test, the HRLMP is collaborating
with laboratories at London Health Sciences
and the Children’s Hospital of Eastern Ontario
to develop NGS-based tests for pediatric and
adult mitochondrial disorders due to nuclear or
mitochondrial genome mutations. HRLMP has
developed and validated two NGS panels for
mitochondrial disorders: (1) 68 nuclear genes
that are associated with adult-onset
mitochondrial diseases, and (2) 37 genes from
the mitochondrial genome. Both panels will be
offered as part of a provincial service beginning
early 2016. By restricting the number of targets
to relevant genes with characterized disease
associated mutations, we have been able to
attain a high depth of coverage across all
clinically relevant regions. This is particularly
important for the analysis of the mitochondrial
genome as disease-associated mutations may
be present in only a small percentage of
mitochondria due to heteroplasmy, and can be
missed against a normal background if there is
insufficient depth of coverage.
The expertise developed through the
hereditary breast cancer and mitochondrial
projects is being directly translated in other
genomics-based projects and analysis of
acquired (rather than inherited) genetic
mutations. A strong collaboration between the
HRLMP and the Juravinski Cancer Centre
Translational Research group has been
particularly fruitful in pushing forward the
development of NGS based companion
diagnostics for solid tumours. Testing of EGFR,
KRAS, NRAS and BRAF is currently available
through the HRLMP as Cancer Care Ontario
funded single analyte assays for specific
tumours. Actionable mutations are assessed in
each gene to predict patient response to
targeted therapies. Conversion of the singleanalyte assays to NGS mutation panels enables
streamlining of work flow and expansion of the
available testing to include a broader range of
clinically useful mutations. The potential for
applying NGS technology to fusion gene testing
and liquid biopsies is also being explored. This
is an important endeavour for the laboratory as
it allows further promotion of an already strong
cancer genetic program and marks increasing
emphasis on “personalized genomics”.
Traditional cytogenetic and molecular genetic
approaches continue as important tools for the
investigation of both heritable and acquired
genetic mutations. However, incorporation of
NGS technologies into the armamentarium
ensures that the HRLMP Genetics Laboratory is
keeping up with the rapid pace of new age
genomics.
Dr. Elizabeth McCready, PhD, FCCMG, Genetics
Dr. John Waye, PhD, FACMG, Genetics
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News from HRLMP
Education News
News from HRLMP Administration
The HRLMP’s 8th Annual Rapid Fire Showcase
was held on Saturday November 21st, 2015 and
was a great success again this year. There were
over 100 attendees both internal to the HRLMP
and external from within our LHIN and beyond.
Twelve members of the HRLMP team, spanning
MLAs, MLTs and management, presented
eleven short oral presentations during this halfday education session. These presentations
covered the wide breadth of the great work
done within our HRLMP laboratories and were
very well done. Thank you to all our speakers
for an excellent morning of continuing
education. The HRLMP is already looking
forward to offering our 9th Annual in 2016!
The HRLMP is pleased to announce that our
Tumour Bank was recognized for the fiscal year
2014/15, and for the third year in a row, as the
OTB Collection Centre with the highest number
of tumours collected. Our Tumour Bank was
also recognized as having the most total donors
and having the fullest sets of tumours with
matching blood samples across the Province.
Congratulations to our Pathology team and
especially our Pathology Assistants for this
outstanding accomplishment!
It is with great sadness that we announce that
Mr. Bill Craig passed away November 22, 2015.
Bill was a valued member of our Management
team and joined the HRLMP in 2011 as a
Supervisor, Hamilton General Core Laboratory,
followed by an administrative role in Special
Projects in 2014. Prior to joining the HRLMP,
Bill had a well-established career in laboratory
medicine. We are very fortunate to have
worked with him.
The HRLMP is excited to be involved in an
international study that has been accepted
(with minor revision) to the Journal of Clinical
Microbiology. This study was collaboration at
ECCMID of laboratories from the Netherlands,
Italy, Canada (Quebec and Ontario (HRLMP))
and the USA. The paper describes the
automated image analysis of chromogenic agar
plates compared to a technologist manually
reading a captured image of the plate on a
monitor using WASPlab image analysis
software.
Mr. David Langstaff, Executive Director, CoLabs
Laboratory Medicine Network, stated
“Congratulations to the entire team for leading
this groundbreaking work … it’s never easy
being on the leading edge, but at times so
rewarding!”.
December 2015  Issue 139
News from Genetics
test will be launched in late 2015 or early 2016.
Myeloproliferative neoplasms (MPN) represent
a rare group of bone disorders in which one or
more blood cell type is overproduced. Affected
individuals have an increased risk for
transformation to acute myeloid leukemia.
JAK2 and MPL have emerged as important
biomarkers in three forms of MPN:
polycythemia vera (PV), essential
thrombocythemia (ET) and primary
myelofibrosis (PMF). Mutations of these genes
are identified in approximately 60% of cases
negative for BCR-ABL1 fusion genes. Until
recently there was little information about the
molecular genetic events that drive the
neoplastic process in BCR-ABL1 negative MPN
cases that also lack JAK2 or MPL mutations.
In 2013 whole exome sequencing efforts lead
to the discovery of acquired (somatic)
mutations in a new gene calreticulin (CALR)
responsible for previously unexplained cases of
MPN (Nangalia et al, N Engl J Med.
2013;369:2391-2405; Klampfl et al, N Engl J
Med. 2013;369:2379-2390). While CALR
mutations are extremely rare in PV (Broséus et
al, Blood. 2014 124(26):3964-6), they are
detectable in approximately 50-85% of
patients with ET or PMF and no JAK2 or PML
mutation (Nangalia et al., 2013; Klampfl, 2013;
Rotunno et al, Blood. 2014; 123(10):1552-5).
The HRLMP Molecular Oncology Laboratory is
currently completing validation of CALR testing
for the investigation of cases with a clinical
suspicion of ET or PMF and absence of
detectable JAK2 mutation. Adding CALR to our
testing menu will increase diagnostic yield in
MPN to up to 90%. It will also provide valuable
prognostic information for MPN patients, as
CALR mutations have been shown to be
associated with different disease course in ET
and PMF and better survival rates in PMF
compared to JAK2 and MPL positive or triplenegative patients(Rumi et al, Blood. 2014
14;124(7):1062-9; ; Blood. 2014, 123(10):154451). It is anticipated that the CALR molecular
Dr. Elizabeth McCready, Genetics
Dr. Daria Grafodatskaya, Molecular Genetics
Fellow
We are pleased to announce that Kristen
Wagner (MLT, Genetics) received the Margie
Patterson Memorial Scholarship in Molecular
Genetics from the Michener Institute’s
Genetics Technology Program. The annual
award is for the new graduate of the program
who has demonstrated excellent technical skills
in the laboratory, consistently exhibited
professional behavior in both academic and
health care settings, has achieved excellence in
their Molecular Genetics studies, demonstrated
leadership skills, shown a commitment to
improving the lives of others, and illustrated
the ability to work effectively both as an
individual and as part of a health care team.
The award was established in memory of
Margie Patterson who passed away in 2005 and
was a long serving technologist in genetics at
HRLMP (see Hemoglobin 2006; 30:1-2). Past
recipients include Robyn White (2007) and
Anna Haasen (2010), both current members of
the HRLMP genetics staff.
Dr. John Waye, Genetics
Genetics Staff Changes
The HRLMP Genetics Laboratory is happy to
welcome Dr. Brenda Murphy to the
professional staff. Dr. Murphy brings with her
considerable experience in clinical laboratory
genetics service and was employed as a
certified genetics technologist for several years
before returning to academia to pursue
graduate studies. After completing her
doctoral degree, Dr. Murphy continued to
demonstrate a strong interest in medical
genetics and completed training in clinical
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molecular genetics through a joint Canadian
College of Medical Geneticists (CCMG)
fellowship training between McMaster
University, Hamilton Health Sciences and Credit
Valley Hospital. She continued to be involved in
Genetics and was responsible for teaching
several courses with the undergraduate science
program at Western University. In 2014 she
completed additional CCMG Cytogenetics
fellowship training at McMaster University and
had been working as a Laboratory Scientist at
Trillium Health Partners – Credit Valley
Hospital. She joined the Genetics Laboratory in
the Summer of 2015 as the Head of Cancer
Cytogenomics and has been a positive addition
to this service.
The laboratory also welcomes Kristen Wagner
as a permanent full-time technologist in the
laboratory. Kristen is an awarded Genetic
Technologist who has recently completed
training through the Michener Institute(see
article above). Her contributions have been
invaluable in the Cytogenetics Laboratory.
News from Hematology
The times are definitely changing! For over 35
years, the smiling face of Wendy Patterson has
become synonymous with excellence in
morphology in the HRLMP’s Malignant
Hematology laboratory at the Juravinski site. In
November of this year, Wendy decided to hang
up her lab coat for the final time and join the
ranks of the retired. Although we are still a
little shell shocked and miss her greatly, we
wish Wendy well in her new life as a retiree,
knowing full well that she will be successful in
that endeavor (and will be able to finally sleep
in after staying up late to watch the Jays and
the Leafs play overtime/extra innings).
With mixed emotions, the Genetics Laboratory
also announces the retirement of a Molecular
Genetics technologist, Ming Ye. While Ming
will be missed in the laboratory, we wish her all
the best in her retirement.
Dr. Elizabeth McCready, Genetics
A celebration tea was held Friday November
13,2015. Thanks to the many that showed up to
wish her well!
December 2015  Issue 139
NEW Eosin-5-Maleimide (EMA) Test for
Diagnosis of Hereditary Spherocytosis
A normal red blood cell (RBC) is shaped as a biconcave disc which allows the red cell to
deform without damage in small capillaries and
within the reticuloendothelial system, including
the spleen. Hereditary spherocytosis (HS) is
due to an inherited abnormality in the RBC
cytoskeleton that causes RBCs to be spherical in
shape. RBCs in HS lose their ability to deform
and are therefore damaged in the narrow
sinusoids of the spleen, resulting in a chronic
hemolytic process.
For the past 100 years the primary laboratory
test for HS has been the Osmotic Fragility (OF)
Test. OF measures the resistance to hemolysis
of RBCs exposed to hypotonic solutions. If a
patient’s RBCs are spherocytic, then they will
be more fragile in higher salt concentrations
compared to normal shaped RBCs, and thus
have an increased OF. Although the OF test is
very sensitive to spherocytes, it is not specific
for HS, and is positive for other forms of
spherocytosis (e.g. warm autoimmune
hemolytic anemia). Furthermore, the results of
OF are not reliable unless samples are kept cold
throughout transport and tested within a
twenty four hour time period. It is for these
reasons the HRLMP has chosen to implement a
new Flow Cytometry test for HS known as the
Eosin-5-maleimide (EMA) test.
In patients with HS, spherocytes have
decreased binding of the EMA dye to
cytoskeletal proteins, and small decreases can
be detected by flow cytometry. For each test
case, a batch of 6 normal controls must be run
and averaged. A decrease in signal that
exceeds established parameters is considered a
positive result. A few very rare disorders may
show reduction in EMA binding, and recent
transfusions may affect results.
This is a very positive process improvement for
the HRLMP to eliminate a test that has been
frustrating due to its labour intensive nature
and lack of specificity as a measure of disease.
The new EMA test is more easily standardized
with increased sensitivity and specificity and
can be turned around in a few hours, and will
be more helpful in the accurate diagnosis of
haemolytic disorders.
Sample case of EMA testing for hereditary
spherocytosis
Dr. Madeleine Verhovsek, Hematology
Dr. Cathy Ross, Pathology
With thanks to Dave Kimmel, Technical
Specialist, Malignant Hematology and Linda
Halchuk, Senior Technologist, Molecular
Hematology and Red Cell Disorders
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Congratulations to Dr. Deborah Siegal, Clinical
Scholar, Division of Hematology and
Thromboembolism, and Dr. Mark Crowther for
their recent publication in the New England
Journal of Medicine of their study on improving
the safety and decreasing the risks of new
blood thinners. The study has successfully
tested an antidote that reverses the effect of
two new blood thinners, Apixaban and
Rivaroxaban.
To read this NEJM free text article, click on the
link below:
http://www.nejm.org/doi/pdf/10.1056/NEJMo
a1510991
To read the Hamilton Spectator press release,
click on the link below:
http://www.thespec.com/news-story/6111530antidotes-mean-more-patients-can-take-bloodthinners/
Congratulations to Dr. John Kelton and Dr. Ted
Warkentin for recently receiving the 2015 Prix
Galien Canada Award, the highest award for
Canadian scientists who have made significant
advances in pharmaceutical research. Drs.
Kelton and Warkentin have worked for more
than 30 years in the diagnosis, characterization,
treatment, and treatment strategies for
heparin induced thrombocytopenia (HIT).
To read more on this outstanding
accomplishment, please click below:
http://dailynews.mcmaster.ca/article/best-inthe-nation-warkentin-and-kelton-to-receiveprix-galien-canada-award/
Save the Date – Friday May 13, 2016 –
5th Multidisciplinary Hematology
Education Day
This educational day, to be held at the
Juravinski Hospital, in the 4th floor Auditorium,
provides an opportunity for Physicians, Nurses,
Pharmacists and Allied Health Professionals to
interact with colleagues who have expertise in
treating patients with Multiple Myeloma,
Myelodysplastic Syndromes/AML, Lymphoma
and CLL. MOCOMP Section 1 credit will apply
and a Certificate of Attendance and hours will
be provided.
December 2015  Issue 139
The HRLMP is pleased to announce that Karen
Moffat, Technical Specialist, Coagulation, has
accepted the role of Chair, Hematology
Scientific Committee, Institute for Quality
Management in Healthcare, effective January 1,
2016. Congratulations Karen!
News from Pathology
We are pleased to inform you that our Breast
Assessment Centre at the Juravinski Hospital
has won the 2015 Cancer Quality Council of
Ontario Quality Award.
Congratulations to the team for this
outstanding accomplishment!
To read the full article, please click on the link
below:
http://www.cqco.ca/awards/award_recipients_
2015/#.Vk6G95fUmTU.gmail
Canada’s first full-time female forensic
pathologist, Dr. Rao, has retired after 31 years
and 7700 autopsies. Dr. Rao’s contributions to
forensic pathology will be remembered fondly
by both the medical and the legal communities.
Quality News
Update from the Pre-analytical Quality
Management (QM) Team
The Pre-analytical QM Team was formed in
September with the aim to address preanalytical gaps identified in our last IQMH (OLA)
assessment. Our first project has been to try
and ensure that Meditech mnemonics (HHS)
or NU identifiers (SJH) are consistently
recorded on specimen labels, along with the
time of collection.
This is being done by a multi-faceted
educational program with audit and feedback
to clinical educators based on specimen
comments being added by HRLMP laboratory
staff. The following comments are being added
to specimens which have missing information:
NOTIME = NO COLLECTION TIME
NOCOLLID = NO COLLECTOR ID
NOTIMEID = NO COLLECTION TIME AND
COLLECTOR ID
ILLID = ILLEGIBLE
“Mom of the Morgue” – Dr. Chitra Rao
The following article recently appeared on the
front page of the Hamilton Spectator: the
HRLMP is proud of Dr. Rao's many
accomplishments;
http://www.thespec.com/news-story/6160531the-mom-of-the-morgue-canada-s-first-fulltime-female-forensic-pathologist-is-retiring/
Thank you to all MLAs and MLTs who have
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taken on the extra work of adding these
specimen comments. An additional thank you
to Mary Kokoski who has supplied us with audit
data and to Nikki Sjoblom who has tirelessly
worked to do individual education with nurses.
We would also like to thank Dan Perryman,
Ronda Snowdy and Audrey Stewart for
providing clinical education, on behalf of the
lab program, for nurses at the Annual Review
for the Juravinski site this month.
Cheryl Main
Allahna Elahie
Cathie McCallum
Research News