Download is known to occur during tumour development,

PROFESSOR SABINE MAI
Nuclear
remodelling of telomeres
Professor Sabine Mai discusses a collaborative project that seeks to understand the mechanisms at
play in the hitherto uncharted 3D nuclear space during normal to cancerous cellular transformation
is known to occur during tumour development,
however it is never studied as a function of
cellular transformation in 3D nuclei of primary
cells from the same lineage.
3D telomere profiling (3D-TP) is a powerful
platform technology that provides a unique
measure of genomic instability applicable
across a wide range of malignancies. At what
stage is 3D-TP in its development, and how
does it measure genomic instability?
To begin, could you describe how you came
to study the mechanisms and consequences
of three-dimensional (3D) nuclear
remodelling of telomeres in cancer?
Our work has focused on genomic instability
and on mechanisms that initiate and/or drive
that instability. We soon realised that there are
very early changes in nuclear structure that
were associated with the development of a
cancerous phenotype and that we therefore
needed to focus on the interphase nucleus and
the remodelling of its nuclear architecture.
When reviewing the literature, it became
apparent that there was not a unified way
of looking at nuclear changes in cancer and
that multiple cell lines and endpoints had
been used. We decided to dedicate our efforts
to primary, immortalised and tumour cells
(not cell lines) of the same lineage to truly
understand what was happening in the 3D
nuclear space during cellular transformation
from normal to cancerous. To define what is a
‘normal’ nuclear architecture we chose to study
telomeres which are the ends of chromosomes.
The rationale behind this was that telomeres
can be used to ‘mark’ chromosome positions in
the interphase nucleus. Telomere dysfunction
We have just started to explore the scope of
the potential of the 3D teleomere analysis in
cancer diagnosis, prognosis and monitoring.
Our group and many collaborating partners
within Canada and abroad have applied this
3D telomere profiling technology in numerous
different tumour settings: from Hodgkin’s
lymphoma to prostrate and thyroid cancer
and acute myeloid leukaemia. Ongoing studies
focus on breast cancer, oesophageal cancer and
cholangiocarcinoma. We are anticipating that 3D
telomere analysis will be a platform technology
applicable across many malignant and
premalignant processes. TeloView, a computer
program for 3D telomere analysis, has been
automated in collaboration with Applied Spectral
Imaging and we are currently performing 3D
telomere scanning of 15,000 cells per hour. We
are now planning the clinical trials needed for
validation of 3D-TP as a clinical biomarker.
How might measurements of genomic
instability applicable across a wide range
of malignancies, sample types, and clinical
scenarios predict biologic activity of a given
malignancy and guide cancer management
in a patient-specific manner?
3D-TP will define the level of genomic
instability that is characteristic for malignancy
and changes as the disease progresses. Once
trials are complete, we hope to be able to use
3D-TP to better define the risk of progression
and likelihood of response to therapy in
patients at diagnosis and develop treatments
appropriate to the biology of the malignancy.
Could you describe the process involved in
obtaining 3D-TP stratified patient samples?
One of the major strengths of this technology
is that the 3D-TP testing can be performed on
a variety of sample types including tumour
biopsies, blood and buccal smears. Formalynembedded and fresh frozen samples can also
be used. After removal of paraffin or fixation of
cells, the telomeres are labelled with a florescent
probe. The microscope is equipped with an
automated image capture system (Telo Scan)
which is able to image over 15,000 individual
nuclei per minute. Analysis of the data generated
then produces a 3D image of the individual cells
and cell populations within the nucleus.
What are the challenges associated with
integrating genomic technologies into
clinical practice?
Taking a laboratory observation and integrating
it into clinical practice is a complex process that
includes many cycles of bench to bedside and
back to bench. Our initial observations must
be confirmed in independent patient cohorts
and then demonstrated to guide treatment in a
way that is independent of current established
prognostic testing. This process involves
collaboration between clinicians, scientists,
healthcare analysts and policy makers.
Have you forged international partnerships
to implement 3D-TP? How important is
collaboration to your work?
Collaborations are key. Without the
multidisciplinary skills the different members of
our team bring to the table, this work could not
have been as successful as it is. Importantly,
now at the verge of clinical diagnostic trials,
the expertise of all clinical collaborators is
advancing the work, its depth and speed.
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PROFESSOR SABINE MAI
Novel 3D cancer genomics
A team at the Manitoba Institute of Cell Biology, University of Manitoba, is investigating how novel
3D telomere profiling can radically change cancer screening, diagnosis and prediction of prognosis
RECENT TECHNOLOGICAL ADVANCES
are having a significant impact on many
areas of cancer research. Cancerous cells are
marked by genomic instability and one of
the most powerful platforms for current hitech development lies in this area. A research
group at the Manitoba Institute of Cell
Biology, headed by Dr Sabine Mai, is using 3D
quantitative fluorescent in situ hybridisation
(FISH) and next-generation sequencing (NGS)
in the hope of optimising this technology for
practical clinical use in cancer treatment.
be separated according to increasing degrees
of genomic instability, which has correlated
with the progression of malignancy”. Moreover,
integrating the 3D-TP measures of instability
with NGS will enable the researchers to analyse
the genomic changes associated with progression
in instability. By identifying and categorising
patients into levels of genomic instability before
performing NGS, the team will increase the
chances of identifying driver mutations.
Telomeres are repeating DNA sequences at the
ends of chromosomes that are involved in the
replication and stability of DNA molecules. On
the realisation that the 3D telomere organisation
of normal cells differed from that of tumour
cells, Mai and her team set about developing
tools that would be able to quantify these
differences. In collaboration with Dr Yuval Garini,
a Professor of Physics and Nanotechnology
at Bar Ilan University in Israel, they created
TeloView, a computer program that measures
the parameters that define variations between
normal and tumour cells.
As part of their research programme, the scientists
are seeking to address the unique diagnostic
challenges associated with prostate cancer,
plasma cell dyscrasias and Hodgkin’s lymphoma.
Crucially, by using 3D-TP, this will be the first
time researchers have tackled these difficulties in
relation to these three types of cancers.
“These measurements include telomere
numbers, telomere sizes, telomere positions
during the cell cycle, telomere distances from the
nuclear periphery and from the nuclear centre,”
Mai explains, the combined results of which
provide a quantitative tool for determining
whether a cell is normal or cancerous. The
benefits of this instrument are many, one
of which is the early detection of cancer, as
she asserts: “If specific profiles are detected
that correlate with early onset, moderate or
severe disease, treatment regimens can be
attempted early on to slow down or prevent the
development of full blown disease”.
3D TP ANALYSIS
Telomere dysfunction is known to occur
during tumour development, and can result in
enormously elevated rates of chromosomal
alterations. To determine the position of
chromosomes, the group tag the telomeres in
nuclei and then image them in 3D using a process
similar to that of CT scans, but at 200 nm steps
through each nucleus. 3D images of the nuclei
are subsequently rendered to display the results
observed in the nucleus, and this is known as 3D
telomere profiling (3D-TP).
3D-TP analysis is highly advantageous because,
as Mai further elucidates: “Clinical material can
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INTERNATIONAL INNOVATION
ADDRESSING DIAGNOSTIC CHALLENGES
Prostate cancer poses a diagnostic challenge
because not all patients with this disease need
treatment. Indeed, men with non-aggressive
prostate cancer can live for years with no
negative impact on quality of life. Careful
monitoring of prostate cancer may be all that
is required, rather than surgery and treatment
with drugs, which can lead to unwanted and
severe side-effects. Thus, there is a need for a
novel diagnostic process that can determine
whether a patient would benefit from watchful
waiting or prostatectomy and radiation
therapy, both of which can cause impotence
and/or incontinence.
In collaboration with clinicians Drs Drachenberg
and Saranchuk (CancerCare Manitoba), the
team is evaluating the effectiveness of 3D-TP
for this type of diagnostic decision-making. Mai
asserts: “We postulate that the tumours with
the greatest genomic instability as measured
by 3D-TP will have the most aggressive disease
and the group of patients demonstrating this
should receive treatment earlier”. Indeed,
categorising patients in this way would mean
that diagnoses are far more accurate and could
potentially lead to a personalised approach to
the treatment of the disease.
PLASMA CELL DYSCRASIAS
AND HODGKIN’S LYMPHOMA
In cases of plasma cell dyscrasias there is currently
no way to predict the risk of a patient progressing
from monoclonal gammopathy of undetermined
significance (MGUS) or smouldering myeloma
(SMM) to multiple myeloma (MM). Working
with clinical collaborator Dr Ade Olujohunbghe
(CancerCare Manitoba), the researchers are able
to define specific profiles for each patient group,
allowing them to detect and develop transition
FIGURE 1. Classical Hodgkin’s lymphoma of mixed cellularity type, LMP1 positive. Bi-nuclear Reed-Sternberg cell surrounded by a corona of lymphocytes. Nuclear DNA of lymphocytes (DAPI, blue) contains multiple small to mid-sized telomeres (red), whereas the central nuclei of the Reed-Sternberg cell are free of telomeres and also show DNA free holes; this
cell is a real, end-stage cancer cell, a ‘ghost-cell’unable to undergo further mitoses. Image provided by Dr Hans Knecht.
INTELLIGENCE
MECHANISMS AND CONSEQUENCES OF
THREE-DIMENSIONAL (3D) NUCLEAR
REMODELLING OF TELOMERES IN
CANCER
OBJECTIVES
To research the primary, immortalised and
tumour cells (not cell lines) of the same
lineage to truly understand the mechanisms
at play in the 3D nuclear space during cellular
transformation from normal to cancerous.
KEY COLLABORATORS
These include:
Dr Yuval Garini • Dr Yvon Cayre •
Dr Francis Wiener • Dr George Klein •
Dr Marie Henriksson • Dr Nir Katzir •
Dr Konrad Huppi • Dr Marc Tischkovitz •
Dr Fabio Morato de Oliveira • Dr Anu Tamm
Dr Marie Punab • Dr Vera Cappelletti
From Canada:
FIGURE 2. Nuclear staining with DAPI (blue) highlights chromatin organisation in the Reed-Sternberg cell of Hodgkin’s
lymphoma, imaged with super resolution microscopy. Image provided by Christiaan Righolt, PhD student in Dr Mai’s lab.
profiles that are presently undeterminable by any
other method.
Similarly, although the majority of Hodgkin’s
lymphoma is treatable, 20 per cent of patients
with this cancer do not respond to treatment and
will die of their disease. Mai explains: “One of the
challenges with Hodgkin’s lymphoma is that the
malignant cell population is a minor component
of the tumour mass and there has not been a
method to study the nuclear stability in the
clinic”. Working with clinical collaborator Dr Hans
Knecht from the University of Sherbrooke and
Dr Donna Wall from CancerCare Manitoba, she
states that: “3D-TP can define different profiles
for the malignant Hodgkin’s and Reed-Sternberg
cells within the lymphoma which are predictive
of clinical behaviour”.
This approach allows the group to develop
a personal profile for each patient using the
trial information. Those profiles will then be
categorised according to the stage of Hodgkin’s
lymphoma, in other words how stable,
aggressive and curable it is, and this will have
a considerable impact on the way patients are
treated after diagnosis.
FROM BENCH TO CLINIC
The ultimate aim of the project is to guide
personalised patient management in cancer.
After the first year of validating previous 3D-TP
observations, the second phase of the project
will focus on demonstrating the potential
impact of 3D-TP on personalised management
in the medical world. Integrating such new
genomic technologies into clinical practice can
be challenging. However, the strength of this
investigation lies in the involvement of many
frontline practitioners, and the valued evaluation
of potential end-users in the clinical trial period.
They hope to identify issues that may affect the
clinicians’ acceptance of the tests and are also
aiming to study the likely impact of 3D-TP on
patients and their families in order to provide
qualitative research input on the important
aspects of the ‘cancer journey’.
The ultimate aim of the project
is to guide personalised patient
management in cancer
University of Sherbrooke: Dr Hans Knecht,
Dr Regen Drouin • McGill University:
Dr William Foulkes • CancerCare Manitoba:
Dr Donna Wall, Dr Don Houston,
Dr Rajat Kumar, Dr Ade Olujohunbghe •
Prostate Center, CancerCare Manitoba:
Dr Jeff Saranchuk, Dr Darrel Drachenberg •
University of Manitoba: Dr Kathleen Gough,
Dr Jim Davie, Dr Michael Mowat, Dr Sabine
Hombach-Klonisch, Dr Thomas Klonisch,
Dr Catalena Birek • The Hospital of Sick Kids:
Dr Uri Tabori, Dr Cynthia Hawkins • The
British Columbia Cancer Agency: Dr Kim Chi •
Zeiss Canada: Dr Oliver Prange
FUNDING
Canadian Institutes of Health Research
CONTACT
Mai is confident that the clinical team will push
the project to its final goal. Furthermore, the
researchers have the full support of Diagnostic
Services of Manitoba, a not-for-profit corporation
responsible for all of Manitoba’s public laboratory
services and for rural diagnostic imaging services,
and the Winnipeg Regional Health Authorities.
The key now is to keep the momentum going:
“We have to keep this positive interaction going
at each stage of the project to ensure its full
uptake,” she affirms.
Professor Sabine Mai, PhD
Senior Investigator, Manitoba Institute of Cell
Biology
Professor, University of Manitoba
Director, The Genomic Centre for Cancer
Research and Diagnosis
If the integration challenges are overcome,
the advancements of this technology could
radically change cancer screening, diagnosis
and prediction of prognosis, leading to optimal
management and monitoring. And it is the
potential for progress from ‘bench to clinic’
that excites Mai the most. She envisions 3D-TP
having a considerable impact on the delivery
of healthcare: “If we succeed, we can change
the way patients are diagnosed and treated”.
The team expects to have licensure in Canada,
US and EU with commercialisation, through
licensure and development of a diagnostic testing
company. Notably, they aim to have 3D-TP in
clinics in the next five years.
T +1 204 787 2135
E [email protected]
Manitoba Institute of Cell Biology
CancerCare Manitoba
675 McDermot Avenue, Room ON6026
Winnipeg, Manitoba
Canada, R3E 0V9
www.umanitoba.ca/institutes/manitoba_
institute_cell_biology
SABINE MAI is Professor of Physiology,
Biochemistry and Medical Genetics, Human
Anatomy and Cell Science at the University
of Manitoba. She completed her PhD in
Molecular Biology at the University of
Karlsruhe. She has received grants from CIHR,
CIHR RPP, Myeloma Canada, CancerCare
Manitoba, Terry Fox Research Institute,
Leukemia and Lymphoma Society of Canada,
and CFI LEF among other funding awards.
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