Page 62 - Hematology_ Basic Principles and Practice ( PDFDrive )

34 Part I Molecular and Cellular Basis of Hematology

gene-independent cell response to CRISPR/Cas9 targeting. More- payers, so demonstrating utility and measurable patient benefit of
over, the ability to express Cas9 or related nucleases is not consistent these approaches is of utmost importance.
across cell types. As with every genome-editing technology, off-target Sequencing-based diagnostics will also likely have an increasingly
effects exist, which may lead to editing of the wrong gene. Because important role in nonmalignant conditions, such as blood-clotting
of the ease of use, libraries have been created targeting the entire disorders, in which it will become possible to systematically rese-
protein-coding region of the genome (i.e., exome), as well as many quence all genes in the coagulation cascade, thereby identifying either
noncoding regions of the genome, which will allow scientists to common or highly rare sequence variants that might explain or
perform comprehensive functional genetics. The combination of predict disease. The widespread use of germline sequencing to predict
genomics with CRISPR/Cas9-mediated perturbation provides an disease also raises a large set of ethical questions that must be
unprecedented opportunity to understand the consequences of the addressed, particularly those relating to children and family members
genetic defects encountered in patients. In addition to pointing to of individuals undergoing sequence analysis. Whether whole-genome
new potential therapeutic targets for cancer, screens using these sequencing will become a routine part of health care in the future
technologies hold the promise of identifying genetic predictors of remains to be determined, but it is almost certain that much of the
gene dependency. Such predictors will be key for the translation of current diagnostic approach to medicine will eventually be supplanted
these in vitro approaches to use in the clinic. by DNA-level analysis.

PHARMACOGENOMICS Minimal Residual Disease Diagnostics

The use of the genome to study drug response deserves special Combination therapies using multiple therapeutic agents have been
mention and is the subject of an entire chapter of this book (see a great success in hematologic malignancies. For instance, childhood
Chapter 8). As the cost of genome sequencing continues to fall, it ALL has been associated with very high cure rates for a long time.
will become increasingly feasible to perform population-scale genetic The therapy of multiple myeloma has been transformed over the last
studies to identify genetic determinants of drug toxicity and response. 15 years with the advent of many new therapeutic agents. Both of
Although some examples of such pharmacogenomic markers have these are examples of diseases for which deep remissions can be
been discovered (e.g., genetic predictors of antimetabolite chemo- achieved, and therefore the ability to detect minimal residual disease
therapy), the field awaits truly large-scale, systematic studies of large (MRD) is of increasing importance. Traditional approaches to detect
numbers of patients with known drug response data. MRD include quantitative PCR, as is used for the detection of the
Bcr-Abl fusion in patients with chronic myeloid leukemia undergoing
treatment with tyrosine kinase inhibitors, or multiparametric flow
CLINICAL USE OF GENOMICS cytometry, which is used for MRD detection in multiple myeloma.
Deep next-generation sequencing of T-cell receptors and B-cell recep-
Sequencing-Based Diagnostics and Precision tors is now being used to detect MRD in B-cell and T-cell malignan-
Medicine cies. This sequencing approach targets a limited number of genomic
regions that are involved in VDJ recombination of the T-cell and
B-cell receptors, thus allowing identification of monoclonal B and T
With the falling cost of genome sequencing and streamlined work- cells, which define the malignant tumor cells. Because these regions
flows, next-generation sequencing has entered into routine clinical are sequenced many times over (i.e., with great “depth”), malignant
diagnostic use. Sequencing panels that target the genes commonly clones can be detected even if they occur with a frequency of only 1
5
6
mutated in cancer are available at many academic centers and are in 10 to 10 . The advances in sequencing of very small cell numbers
actively being used to triage patients to specific treatments. Genomic down to the single-cell level, as described earlier, allow for compre-
variants in such a “precision medicine” approach either suggest a hensive detection of genomic alterations as well as interrogation of
therapeutic agent that directly targets the variant itself (e.g., a BRAF transcriptional profiles. Combining these sequencing approaches
inhibitor in a patient with a BRAF V600E mutated neoplasm) or with highly sensitive cell isolation and cell-sorting technology holds
inform therapeutic decisions that are less directly related (e.g., not great promise to better characterize what types of tumor cells remain
using epidermal growth factor receptor inhibitors in colon cancers viable at the stage of MRD.
harboring KRAS-activating mutations). Similarly, genomic variants
may predict an individual’s response to immunotherapy. In particular,
it has been observed that tumors with high mutation burden tend to Expression-Based Diagnostics
have more favorable responses to programmed death 1/programmed
death ligand 1 (PD1/PDL1) blockade than in patients whose tumors It has been over a decade since the first proof-of-principle studies were
have fewer mutations, presumably reflecting a lower neoantigen load. published demonstrating the possibility of using gene expression
Compared with RNA-based analysis, DNA-based diagnostics profiling to classify diseases such as cancer. Those studies raised the
have the advantage of being more definitive in that one is looking, possibility that such promising gene expression signatures might be
for example, for the presence of a mutation (an A, G, C, or T) as further validated and then implemented in the routine clinical setting
opposed to a relative abundance of a particular transcript or tran- as powerful diagnostic tests. The reality is that few such transitions
scripts, the latter being confounded by an admixture of cell types to clinical practice have been made. One of these is the Oncotype
within tumors or tissues. Because modern sequencing approaches Dx Breast Cancer Assay test, which consists of a tumor gene expres-
allow for allele separation, the admixture of tumors with normal cells sion signature of 21 genes capable of determining the requirement
can be addressed at the DNA level simply by increasing the depth of for chemotherapy in women with early-stage breast cancer. This test
sequencing coverage, as described earlier. Whether single-cell RNA has now become part of the standard of care at many cancer centers
sequencing approaches (which combat the issue of cellular heteroge- in the United States.
neity) have the potential to be introduced into the routine clinical One should ask, however, why, despite thousands of papers being
setting remains to be determined. Currently, their technical complex- published on potential diagnostic applications of gene expression
ity precludes routine use. Similarly, if the cost of sequencing continues profiling, so few have progressed to routine clinical implementation.
to drop, this will likely give way to more systematic approaches that There are likely several reasons to explain the slow pace of advance-
include whole exome sequencing and whole-genome sequencing ment. First, to develop truly valid diagnostic tests, the test must be
rather than just sequencing a limited number of loci. However, the applied to large numbers of patients with known clinical outcomes,
pace of technology advancement will likely outstrip understanding of and in many cases, such cohorts of patients simply do not exist,
clinical utility and financial reimbursement by health insurance making validation challenging. Second, because gene expression