Page 1347 - Clinical Immunology_ Principles and Practice ( PDFDrive )

1308 ParT ElEvEN Diagnostic Immunology

Clinically Important Applications of Cell-Free DNA and Liquid Biopsy
Next-Generation Sequencing It has been known for many years that there is normally a small
Molecular diagnostic techniques have a wide range of potential amount of genomic DNA in the cell-free plasma fraction of
applications in clinical immunology. DNA diagnostic proce- blood (called cfDNA). It is thought that this material comes
dures are used to (i) perform HLA genotyping; (ii) analyze from the normal processes of cell apoptosis, particularly from
and monitor neoplastic disease; (iii) provide identification or leukocytes. During pregnancy, there is a significant contribution
DNA “fingerprinting”; (iv) monitor bone marrow engraftment; of DNA from the placenta, and thus cfDNA can be used as a
(v) establish a genetic diagnosis in a symptomatic individual; biological surrogate for the fetal genome. NGS enables sampling
(vi) determine the risk of occurrence of a disease in offspring; of this DNA at very high read depth, and common chromosomal
and (vii) to establish a prenatal diagnosis. The use of DNA imbalances in the fetus can be detected by analysis of DNA
analysis for HLA typing is described in Chapter 5. DNA extracted from maternal plasma. The observation of a patient
techniques are used in leukemias and lymphomas, primarily who had cancer during pregnancy led to the discovery that cancer
for investigation of cell lineage, proliferative clonality, and cells may also contribute to cfDNA. New genomic assays for
measurement of residual abnormal cells after therapy. Molecular detection of cancer recurrence, changes in mutation spectrum,
analysis, and especially molecular cytogenetic analysis, is impor- and clonal expansion are rapidly arriving in clinical practice.
tant in guiding initial and follow-up therapies (Chapters 78 These assays are likely to be especially useful in lymphomas and
and 79). other solid tumors that have required invasive biopsies. Serial
testing by these so called liquid biopsy methods is very likely to
Targeted Panels have a large impact in immune-oncology practice.
When the phenotype is sufficiently circumscribed that a limited
list of genes is known to account for a high percentage of cases, LABORATORY STANDARDS AND REPORTING
it may be appropriate to use a targeted gene panel as the first-
round genetic diagnostic test. The reasons include assay design All US laboratories providing any clinical data are subject to the
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strategy that puts maximum emphasis on complete gene coverage; regulatory requirements embodied in CLIA88. CLIA88 mandates
lower unit cost of testing; and reduced occurrence of secondary biannual laboratory inspections, quality control, quality assurance,
findings (i.e., apparently pathogenic variants in genes not sus- proficiency testing, and personnel training standards. The
pected from the primary indication for testing). But efficient American College of Medical Genetics has also produced guide-
use of targeted panels requires high sensitivity in the sense lines for diagnostic laboratories conducting molecular genetics
that most of the important disease-causing genes must have testing. A clear argument can be made for the clinical utility of
been accounted for and give a conclusive diagnosis in a large early diagnosis for rare diseases, but demonstrating reduced cost
fraction of clinical cases. This situation is often not met because of care or improved clinical outcome is difficult. Genomics tests,
of the lack of specificity in clinical presentation or the very large like all diagnostic testing, should be designed and developed
degree of locus heterogeneity. The latter is particularly important such that the practiced test can meet the requirements of its
in PIDs. intended use. The performance of genomic tests must be validated
before they are offered for clinical diagnostic use. Critical measures
Whole Exome/Whole-Genome Sequencing of performance include sensitivity—how often the test is positive
In the past, clinical genetic testing would necessarily stop after when a disease causing variant is present; specificity—how often
the most likely genes had been investigated in a few targeted the test is negative when a mutation is not present; “technical”
laboratory tests. That strategy changed in the last few years positive predictive value (TPPV)—the fraction of true positives
after it became possible to efficiently assess most known disease divided by the sum of the true positive and false-positive tests;
genes in a single test. Two approaches are in use today: WES and positive and negative percent agreement (PPA) with previous
and WGS. WES depends on “capture” of the 1–1.5% of the reference standards. These are all measures recommended by
genome that contains the protein-coding segments of genes. the FDA in its draft guidance for validating clinical NGS tests.
Capture is typically achieved by liquid phase hybridization New methods to test and validate bioinformatics software are
followed by PCR and sequencing library construction. The also needed as the field moves forward (Fig. 96.7).
rationale for WES is that most interpretable disease causing
variants occur in coding sequence, and thus the sequencing FUTURE DIRECTIONS
is focused on the interpretable fraction of the genome. The
lower unit cost of WES also allows for greater read depth with The next decade will bring further improvements in our ability
concomitantly lower error rates. Some significant limitations to identify family-specific pathogenic variants for all genes that
of WES include incomplete coverage of disease-causing genes cause rare mendelian disorders, such as PIDs. Sequencing technology
because of inefficient capture; uneven coverage across all targeted will continue to develop, further reducing the cost of genome
regions such that adding more read depth inefficiently adds analysis for individuals and families. As our understanding of the
to poorly covered regions; poor ability to resolve structural role of polymorphisms in disease risk increases, the importance
variants; limited ability to call copy number variants; and of low-cost, high-throughput genotyping of standard variant panels
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inability to call triplet repeat mutations. WGS, especially when will also increase. The importance of bioinformatics in data
implemented with PCR-free library construction techniques, analysis will become increasingly apparent as large amounts of
overcomes most of these limitations. The falling cost of WGS, individual sequence data are produced. International clinical variant
especially when coupled to automated library preparation systems, databases will play an important role in both diagnosis and
holds the promise of nearly complete analysis of the genome of prognosis. New statistical approaches will be needed to exploit
individuals. fully the potential of complete genome sequence for estimation