Keywords
molecular - driver mutations - monitoring - minimal residual disease - biomarker
Introduction
In colorectal cancer (CRC), approximately 25% present with metastatic disease and
more than half will ultimately require treatment for metastatic disease.[1] While prognosis remains poor for the majority, a significant number of such metastatic
CRC patients can be long-term survivors with the use of cancer-directed systemic therapy.[2] We use tumor tissue characteristics for prognostic and predictive significance.
Biomarkers are a valuable addition to the armament. We know that early identification
of the high-risk subset among CRC patients is the path toward early intervention that
would lead to better overall survival (OS), save treatment costs, and perhaps cure
a larger portion.
Precision medicine and personalized oncology is dependent on identifying specific
genetic alterations that can identify high-risk subset and enrich the population that
is most likely to benefit from targeted therapy.[3]
[4]
[5] Unfortunately, routine tissue biopsy has several limitations. Some of the well-known
ones include intratumoral heterogeneity, limited tissue sample size, challenge of
accessing difficult sites of disease, patient discomfort, and the inability to repeat
invasive procedures.[6]
Liquid biopsy circumvents most of these problems and has been gaining increasing importance
in several cancers, including CRC. Liquid biopsy is the term used for a diagnostic
sample obtained from a patient's body fluid (most commonly blood) that can be tested
for specific cancer-related aberrations.[7]
[8] Its value and application in the routine care of CRC has gained increasing acceptance.[9]
[10]
[11]
[12]
[13]
Methods
Liquid biopsy for CRC can consist of three types of analysis—circulating tumor cells
(CTCs), circulating tumor deoxyribonucleic acid (ctDNA), and/or circulating noncoding
ribonucleic acids (RNAs) (inside or outside exosomes).[14]
Initial efforts were focused on the identification of CTCs. These could have reached
the blood circulation from either the primary tumor or from a metastatic site. Isolation
of CTCs was based on the principle that they are epithelial cells. Initial studies
were promising. Unfortunately, we now know that CTC assays are difficult to standardize
and are positive in only approximately 30% of patients with advanced CRC.[15] Their concordance with molecular changes in the primary tumor were also limited,
seen in 50 to 70% of cases.[16]
Focus quickly shifted to ctDNA. These could also represent either shedding from the
primary tumor or the metastatic site. But detection techniques were more robust and
consistent—polymerase chain reaction (PCR) or next-generation sequencing (NGS). Concordance
with tumor biopsy samples was also found to be higher.[17] For instance, Yu et al did a study in 150 CRC patients with metastatic disease.
They showed a concordance rate of as much as 92% when plasma ctDNA digital PCR was
compared with primary tumor tissue DNA for common BRAF and KRAS mutations.[18] Some studies have also shown a lower concordance rate, but that is likely to be
due to the two samples having been obtained at different time intervals.[19] Evaluation of three advanced methodologies (COLD-PCR, microarray, and droplet digital
PCR [ddPCR]), for identifying the mutational status by liquid biopsies in metastatic
CRC patients is gaining momentum.
Recent attention has also been on circulating noncoding RNAs. One large study of 326
CRC patients showed good correlation between exosomal miR-21 from liquid biopsy specimen
as compared with that from CRC tumor tissue.[20]
When the strengths and weaknesses of the above three methods are evaluated, ctDNA
is a clear winner. It is well-established and widely studied methods (e.g., NGS, ddPCR,
Amplification Refractory Mutation System [ARMS]), has high sensitivity and consistent
correlation, can predict drug response/resistance, and helps in monitoring of CRC
patients—especially for early detection of progressive disease.
Utility in CRC
Today, it is possible to predict risk of tumor metastasis using genome-wide sequencing
along with bioinformatics tools like biological pathways, receptors, and protein network
as well as artificial intelligence/machine learning. For patients with advanced CRC,
it is the genomic instability and additional molecular alterations occurring at the
metastatic site that determines patient prognosis and guides further therapy.[21]
[22]
[23] One study of 47 CRC patients in early or late cancer stages and found that stage
IV patients had significantly higher ctDNA concentrations than stage I patients.[24] Another study of 97 metastatic CRC patients demonstrated that those with higher
ctDNA burden had shorter OS. This was also true for those with BRAF mutations as compared
with the BRAF wild-type cohort.[25] A third study looking at pretreatment ctDNA confirmed that patients with increasing
levels while on first-line chemotherapy (CT) had poorer outcome.[26] Tie et al showed that falling ctDNA levels (more than 10-fold reduction) after first-line
CT predicted longer progression-free survival (PFS).[27] The same group also showed that in surgically resected patients, if ctDNA was detectable
postsurgery, there was a higher 5-year recurrence risk (38.6% vs. 85.5%) and lower
OS (64.6% vs. 89.4%).[28]
[29] In addition, a recent study in 23 patients of CRC with deficient mismatch repair/microsatellite
instability-high monitored serial ctDNA during programmed cell death protein 1 (PD-1)
blockade. It was successful in predicting responses weeks earlier than standard imaging
studies—thus indicating the role of ctDNA in predicting early tumor response to immunotherapy.[30] In a multicenter study involving 265 patients with nonmetastatic CRC (stage I–III),
serial ctDNA testing was able to detect minimal residual disease (MRD) 8 months earlier
(range, 0.56–21.6 months) than radiologic evidence of relapse.[31] The IDEA-France phase III trial, gave valuable insight into positive ctDNA results
being an independent prognostic marker—in fact it helped select ctDNA positive patients
for longer treatment (6 months) to provide outcome that was similar to ctDNA negative
group (treated for 3 months).[32] Not surprisingly, meta-analysis has confirmed the prognostic and predictive value
of pretreatment ctDNA levels in the management of CRC.[33] Measuring ctDNA has a role in selecting patients that do not require adjuvant CT
as well.[34] This large study included 455 patients with stage II colon cancer, of which 302
were managed based on ctDNA results whereas 153 received standard treatment and were
the control arm. The ctDNA-guided arm received less adjuvant CT (15%) as compared
with the standard therapy arm (28%; relative risk of1.82 with a 95% confidence interval
[CI] of 1.25–2.65). The recurrence-free survival at 2 years was identical in the two
groups (93.5% in the ctDNA-guided arm and 92.4% in the standard arm). This strategy
allows a significant number of stage II colon cancers patients to avoid unnecessary
adjuvant CT without increasing the risk of recurrence. Use of ctDNA for serial RAS
mutational evaluation can help in deciding right rechallenge strategy for selected
patients.[35] A phase 2 single-arm multicenter study involved patients who were previously treated
with irinotecan and cetuximab in the first line as well as oxaliplatin and bevacizumab
in the second line. The ctDNA showed RAS mutations in 12/25 evaluable patients (48%)
at second relapse. When rechallenged with irinotecan and cetuximab, the group with
wild-type RAS mutations on ctDNA had better PFS (median PFS 4.0 vs. 1.9 months).
ctDNA methylation has also been shown to be a good biomarker. One study involving
five methylation genes (ITGA4, EYA4, GRIA4, MSC, and MAP3K14-AS1) predicted better response and PFS.[36] There is also value in quantifying the methylated circulating DNA. For instance,
one recent study showed that the median DNA methylation levels of TMEM240 promoter
hypermethylation for CRC is 0.0021 while it remained undetectable (0.0000) in healthy
subjects.[37] Another study on cfDNA (cell-free DNA) looked at three tumor-specific DNA methylation
markers (chromosome 9 open reading frame 50 [C9orf50], CAP-Gly domain containing linker
protein family member 4 [CLIP4], and potassium voltage-gated channel subfamily Q member
5 [KCNQ5]) showed the ability to distinguish stage in CRC patients with a specificity
of 99%. The sensitivity was 80% for stage I, 85% for stage II, 89% for stage III,
and 88% for stage IV.[38]
[39] Another study involved 299 patients having stage I to III CRC. Of the 296 patients
in whom preoperative samples were available, 232 (78.4%) had tested positive for at
least one of the six ctDNA methylation markers [33a]. One month after surgery, ctDNA-positive
patients had 17.5 times higher risk of relapse (hazard ratio [HR], 17.5; 95% CI, 8.9–34.4;
p < 0.001). After adjuvant CT, ctDNA-positive patients had shorter recurrence-free
survival (HR, 13.8; 95% CI, 5.9–32.1; p < 0.001). And sequential ctDNA analysis showed that ctDNA-positive patients had poorer
recurrence-free survival (HR, 20.6; 95% CI, 9.5–44.9; p < 0.001). Discovery of methylated circulating DNA biomarkers for comprehensive noninvasive
monitoring of treatment response in metastatic CRC is also of value in the follow-up
of CRC patients who do not have driver mutations.
Beyond Circulating Tumor Biomarkers
Beyond Circulating Tumor Biomarkers
This article would not be complete without a mention of use of liquid biopsy for (1)
oral microbiota related bacterial network, (2) gut microbiome-associated serum metabolites,
and (3) P-element Induced WImpy testis (PIWI)-interacting RNAs (piRNAs), microRNA(miRNAs),
long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs).[40]
Saliva and stool matched samples from patients with CRC patients, when compared with
those from healthy controls, have identified four bacterial species found in abundance
predominantly in CRC patients only—they are Solobacterium moorei, Streptococcus anginosus, Streptococcus koreensis, and Peptostreptococcus stomatis
[.41] Other organisms of interest are Fusobacterium
nucleatum and Streptococcus gallolyticus subspecies gallolyticus (Sgg). Measurements of anti-F. nucleatum immunoglobulin A levels could serve as a robust biomarker for CRC and might find
its place in screening high-risk persons in the community.[42] We also know that Sgg promotes the development of CRC. Liquid biopsy measurement
of type VII secretion system (T7SS; called SggT7SST05) is also known to identify persons with colonization by Sgg and hence high risk for
the development of CRC.[43]
Conclusion
Liquid biopsy is quickly cementing its crucial role in the management of patients
with CRC. Its advantages include being of noninvasive nature, ability to do frequent
sampling, help as additional tool in diagnosis, have prognostic and predictive significance
as a reliable biomarker, and guide personalized therapy by monitoring patient disease
status several weeks ahead of other diagnostic modalities. In India and South Asian
countries, their utility shall require access to reliable laboratories that have demonstrated
consistent validation of sample handling/transport, wet laboratory processing, and
robust bioinformatics that allow quick turnaround time. In the future, ctDNA could
also be used as a surrogate endpoint for response rate, PFS, and even OS. If this
concept stands the test of time and proves to be a robust marker, patients would be
the ultimate beneficiary in many ways. We end with a word of caution. If using ctDNA
for MRD results in a significant false negative rate (which has been reported to be
as much as 15% in some publications), it could lead to undertreatment. Similarly,
patients who are false positive could receive additional therapy that was unnecessary.[44] We need to remember that currently ctDNA as a companion diagnostic test is not considered
standard of care. So, if a CRC patient is identified solely by ctDNA to have RAS mutations
or BRAF V600E use of targeted therapy guided by liquid biopsy should be used with
caution.[45]
