TRAINING and EDUCATION
Demystifying the Lab
Continued from page 111
and differentiate malignant and nonmalignant
cells – and to assess their patterns of differ-
entiation. Unlike flow cytometry, immuno-
histochemistry has the advantage of allowing
pathologists to assess the expression of certain
markers in the contest of tissue architecture
and structure, according to Dr. Jaye.
But the important malignant cells might
be few and far between and in the clinical
setting, and immunohistochemistry allows
labeling of one slide with only a single anti-
body. This means that pathologists need to
make qualitative assessments to understand
if a certain cell on one slide is the same type
of cell that is labeled with a different antibody
on another slide. “That can be frustrating,” Dr.
Jaye noted, “but, with flow cytometry, we can
confidently identify the patterns of expression
of multiple antigens on the same cell, remov-
ing the chance of human error.”
Mike Keeney, associate scientist at the
Lawson Research Institute in London, Ontar-
io, Canada, agreed. “Flow cytometry is a more
definitive methodology. Pathologists looking
under a microscope need to evaluate whether
what they are looking at are malignant cells,”
he said. “Flow cytometry gives us an objective
assessment that is not based on an individual’s
judgment.”
The advent of multicolor flow cytometry
actually has decreased the amount of a patient
sample needed for processing. “What’s so
wonderful is that we don’t need more sample
to assess more markers,” Dr. George said. “It’s
quite the opposite: It’s fewer cells for more
information.”
“We only need 1 or 2 milliliters of
cerebrospinal fluid to determine whether a
malignancy has invaded the central nervous
system,” Mr. Keeney added. “This was not
possible before.” Flow cytometry also is allow-
ing pathologists to move away from having
to remove a patient’s entire lymph node to
test for malignant cells, to taking a small, fine
aspirate sample for diagnosis and analysis, in
some cases.
From Diagnosis to Prognosis
Aside from diagnosis, multicolor flow cytom-
etry is used in the clinic for other aspects of
the management of patients with hematologic
malignancies, including prognosis and thera-
py selection. Often, the pathologist is making
a diagnosis, assessing prognostic markers, and
determining which could be used for therapy
selection – all at the same time. For example,
if a diagnostic flow cytometry analysis reveals
that a patient has CD30-positive lymphoma,
the presence of the CD30 marker also in-
dicates that the patient may be eligible for
therapy with brentuximab vedotin, a CD30-
targeting antibody drug conjugate.
The antibodies used to detect malignancy-
specific and nonspecific markers can also be
used for prognosis. Whether a patient with
CD30-positive anaplastic large cell lymphoma
also harbors a rearrangement in the anaplastic
lymphoma kinase (ALK) gene can help clini-
cians with a prognosis, because patients with
ALK-negative anaplastic large cell lymphoma
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ASH Clinical News
have less favorable outcomes and poorer
survival. 6
“We are often making the diagnosis and
looking at prognostic and therapeutic mark-
ers, as well,” Dr. George said. “We select the
markers to analyze on the basis of whether the
patient is suspected to have a lymphoma or
leukemia, for example. Then, based on the ini-
tial results, we may add on additional markers
for more detailed characterization.”
An Emerging Application: MRD Detection
Detection and monitoring of minimal residual
disease (MRD) is now becoming a standard
practice for patients with hematologic ma-
lignancies, including for acute lymphocytic
leukemia, acute myeloid leukemia, and multiple
myeloma. 7,8 MRD refers to the counting of rare
malignant cells that remain after a patient has
completed a course of therapy or is in clinical
remission. MRD typically is assessed via samples
from a blood draw or a bone marrow aspirate.
MRD detection allows clinicians to
evaluate a therapy’s efficacy, can help select
subsequent therapy options, and serves as a
prognostic marker – providing an indicator
of how long a patient’s remission might last,
how deep the remission is, and when and if a
patient is likely to relapse.
Flow cytometry is one of the sensitive
methods used to detect MRD, along with
DNA-sequencing technologies that detect
the DNA unique to malignant cells. Using
flow cytometry to measure MRD sometimes
requires a larger sample than that needed for
other research applications, since the techni-
cian is looking for the likely-rare malignant
cell that could have remained following
therapy, which requires scanning many more
normal cells.
“There are now guidelines for detection
of MRD in patients with certain hematologic
malignancies,” said Dr. George, referring to
the International Clinical Cytometry Society’s
consensus recommendations. 9 There are only
a few labs in the U.S. that represent the “go-to”
experts for MRD detection and serve as refer-
ence labs for other clinical centers. The efforts
to standardize the technique have come from
the flow cytometry community itself. “More
and more, labs want to do MRD-testing, and
it has become clear that, frankly, some labs
weren’t doing this well. Now, we have rigorous
criteria for how to set up MRD-testing at an
institution. We are actually just in the process
of finalizing this at our center,” he stated.
“So far, only a subset of extensively experi-
enced pathologists specializes in MRD detec-
tion because it is so technically challenging,”
Dr. George noted.
For Dr. Jaye, one advantage of using flow
cytometry for MRD detection is that the cell
detection technique, unlike DNA-sequencing
methods, does not require the technician to
know the patient’s original diagnosis. To use
DNA sequencing, clinicians need to look
for specific mutations present in the malig-
nant cells. “But, with flow cytometry, we can
identify malignant cells without knowing the
patient’s diagnosis or the mutations found in
their malignant cells, because we are simply
looking for those cells that are not normal in
the population,” he explained.
New Therapies, New Challenges
One challenge of using flow cytometry for
MRD detection or standard disease-tracking
is working around the effects of newer tar-
geted treatments. For example, if a patient
is treated with an anti-CD30 antibody, the
CD30-expressing cells may no longer be
present following treatment. The diseased cell
population shifts in their marker expression
profile, requiring new antibodies against new
markers for the cells’ detection.
“When clinicians treat a multiple my-
eloma patient with an anti-CD38 therapy, for
example, that results in loss or masking of the
CD38 antigen,” Dr. Jaye explained. “So, when
we analyze a post-therapy biopsy, we will no
longer have CD38 in our toolbox to recognize
the malignant cells by flow cytometry.” Scien-
tists and pathologists now are trying to find
the right combination of novel markers for
post-treatment flow cytometry analyses.
Another challenge for MRD detection – in
which technologists look for the rare “needle-
in-the-haystack” diseased cell – is sample
quality. “Bone marrow samples are, by nature,
contaminated with peripheral blood cells, but
some samples will be ‘bloodier’ than others,”
said Dr. George. These contaminated samples
make the detection of rare malignant cells
especially challenging. “I’ve been a pathologist
in a number of centers, and the quality of the
sample is the biggest issue with flow cytometry
everywhere. If it’s garbage in, it’s garbage out.”
There is also a lack of skilled pathologists
and flow cytometry technicians who can per-
form diagnosis and follow-up of patients with
hematologic malignancies, according to Dr.
George. “It’s a big problem in the lab indus-
try,” she said. —By Anna Azvolinsky ●
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