Written in Blood
needed to assess defibrotide in
combination with other therapies
and in high-risk patient groups,
such as those who are exposed to
sirolimus and VOD/SOS prophylaxis for patients undergoing
allogeneic or high-risk autologous
HCT.
o 8% B-cell lymphoma not
otherwise specified
REFERENCE
o 8% mantle cell lymphoma
Richardson PG, Riches ML, Kernan NA, et al. Phase 3 trial of
defibrotide for the treatment of severe veno-occlusive disease
and multi-organ failure. Blood. 2016;127;1656-1665.
Novel DNA/RNA Genomic
Profiling Assay Identifies Broad
Range of Genomic Alterations in
Hematologic Malignancies
Researchers have developed a
novel, next-generation sequencing-based assay that successfully
characterized genomic alterations
in 93 percent of patients with
hematologic malignancies. This
genomic-profiling approach has
the potential of increasing the
ability to identify clinically and
therapeutically relevant alterations, according to Jie He, PhD,
from Foundation Medicine in
Cambridge, Massachusetts, and
colleagues, whose findings were
published in Blood.
“Cytogenetic studies have
identified recurrent mutations
and amplifications/deletions
in a spectrum of hematologic
malignancies, creating a pressing
need to develop comprehensive genomic assays to identify
somatic alterations,” Dr. He and
co-authors wrote. “However,
current diagnostic assays, including FISH and real-time PCR,
are designed ad hoc to identify
specific genomic alterations, and
in some cases there are no assays
that can reliably identify specific
rearrangements.”
The newly developed assay
uses targeted next-generation
DNA and RNA sequencing to
identify single-nucleotide substitutions, insertions and deletions,
copy number alterations (CNAs),
and rearrangements.
To test the accuracy of this
assay, Dr. He and researchers
first extracted DNA and RNA
from each of the 3,696 patients,
categorized them into barcoded
libraries through separate workflow streams, and then pooled
and sequenced the samples. The
investigators then performed
blinded comparisons between
the novel assay and DNA-only
diagnostic assays, including
real-time PCR, FISH, and PRC
fragment analysis, for a large
number of clinical samples. The
38
ASH Clinical News
concordance between the two
sets of results was 99.4 percent,
“[demonstrating] that the current DNA profiling platform can
accurately detect DNA sequence
variants and CNAs, as established for the reference assay,”
the authors reported.
The novel assay was also
evaluated for its ability to
detect genomic rearrangements. Sensitivity and specificity (determined by comparing
the genomic rearrangements
detected in the pooled samples
relative to the constituent celllines) were high (at 100% and
98%, respectively), and very
few false-positive responses
were observed, with a positive
predictive value of ≥98 percent
(n=245/248). Results from the
novel assay were reproducible
between batches, as well.
The new assay has been used
to perform genomic profiling in
3,696 patient samples, and 3,433
(93%) were successfully characterized via DNA and RNA, including:
• 27% formalin-fixed paraffinembedded samples
• 21% bone marrow aspirates
• 18% blood samples
• 34% samples from preextracted nucleic acids from
relevant tumor tissues
The specimens submitted came
from patients with the following
diagnoses:
• 39% multiple myeloma
• 22% non-Hodgkin lymphoma
o 41% diffuse large B-cell
lymphoma
o 13% follicular lymphoma
• 17% acute myeloid leukemia
• 13% myelodysplastic
syndromes/myeloproliferative
neoplasms
“We showed that a single, integrated assay can be used to find
the spectrum of clinically relevant
disease alleles, including mutations, translocations, and gains
and losses,” Ross Levine, MD, a
co-author of the study, told ASH
Clinical News. “In ma ny cases,
having this information is of value
with respect to clinical care.”
Extracted DNA was sequenced
to an average depth of 500x and
RNA to an average of 6.9 million
unique pairs. Seven percent of the
specimen (n=263/3,433) did not
attain quality control specifications, including failures in DNA
or RNA preparation, or blood
samples older than acceptable criteria with no evidence of disease,
or no somatic driver mutations
reported under the study’s qualifying conditions.
“Genomic profiling of hematologic malignancies demonstrates that only a relatively small
number of genes are commonly
mutated while many specimens
harbor a wide range of rarer
events, including base substitutions, indels, copy number amplification/losses, and rearrangements,” the authors reported.
At least one driver mutation
was identified in 95 percent of tumor specimens (n=3,246/3,433),
77 percent (n=2,650) of which
harbored at least one alteration
linked to an approved targeted
therapy or one in clinical trials,
including:
•
•
•
•
•
•
•
NRAS (14%)
KRAS (13%)
DNMT3A (7%)
CDKN2A (7%)
IDH1/2 (5%)
BRAF (4%)
FLT3 (4%)
In 61 percent of cases, at least one
alteration with known prognostic
relevance in a particular tumor
type was identified, including:
•
•
•
•
•
TP53 (19%)
ASXL1 (9%)
TET2 (8%)
CDKN2B (5%)
CREBBP (5%)
• MLL (4%)
• NPM1 (2%)
A total of 1,524 genomic rearrangements were identified in 37
percent (n=1,256/3,433) of tumor
specimens, involving genes implicated in:
• Chromatin/histone remodeling
(20%)
• Transcriptional regulation
(16%)
• Kinase/oncogene activation
(15%)
• Apoptosis regulation (13%)
• Cell cycle regulation (13%)
• Truncation of tumor
suppressors (5%), including
novel in-frame fusions in kinase
drug targets in ALK, BRAF,
FLT3, JAK2, and ROS1
Dr. He and researchers were
also able to identify genomic
alterations in different patients
that activated specific subsets
of oncogenes, such as somatic
alterations involving the FLT3
locus that have been shown to be
of prognostic importance in acute
leukemia. Eighty-nine alterations
involving FLT3 in 84 specimens
were found, as well as other base
substitutions (n=14), in-frame
deletions (n=1), CNAs (n=6), and
fusions involving FLT3 (n=1),
“indicating that the sequencingbased assay can robustly detect a
wide range of alterations in driver
genes that are not fully evaluated
using conventional methods.”
“The assay is rapid, comprehensive, accurate, and able to find
lesions in DNA and RNA,” Dr.
Levine told ASH Clinical News.
“This allows us to offer genomic
testing to a broader set of patients
with blood cancers.”
The study was limited by RNA
preparation failure that occurred
in 132 cases, thus indicating a
challenge of processing and sequencing RNA samples.
Dr. He, the study’s first
author is an employee of Foundation Medicine, a company that
develops, manufactures, and sells
genomic analysis diagnostics. ●
REFERENCE
He J, Abdel-Wahab O, Naha MK, et al. Integrated genomic
DNA/RNA profiling of hematologic malignancies in the
clinical setting. Blood. 2016 March 10. [Epub ahead of
print]
May 2016