Prevalence of clonal hematopoiesis may confound tumor-only genomic profiling results

As the co-chair of this year’s ASCO Tumor Biology Oral Abstract Session in Chicago, I was particularly looking forward to a portion of our 3-hour session on Tuesday, June 5, dedicated to the issue of clonal hematopoiesis and its potential impact on liquid biopsy results for patients with cancer. Clonal hematopoiesis is a hot topic because mutations coming from white blood cells (WBC) that shed their DNA into the blood can potentially confound results of liquid biopsy tests and can potentially mislead clinicians with their interpretation of the results. This can have a negative impact on care of patients if they are deprived from potentially effective therapy by erroneously concluding that a drug resistance mutation is present. A good example of this is if a patient with colorectal cancer is found to have a KRAS mutation on liquid biopsy testing, they may be deprived of potentially effective anti-EGFR therapy.

Two studies were presented, one by Charles Swanton, MD, PhD, who summarized data using the GRAIL platform that is working on a cell-free DNA Cancer Genome Atlas project for early cancer diagnosis, and the second by Ahmet Zehir, PhD, which focused on the prevalence of clonal hematopoiesis mutations when tumor genomic tests were performed using tumor tissue only. Both of the presenters found evidence for mutations in the blood attributable to clonal hematopoiesis derived from WBC. Clonal hematopoiesis occurs more frequently with aging and is sometimes referred to as age-related clonal hematopoiesis as described by Kenna Mills Shaw, PhD, from The University of Texas MD Anderson Cancer Center, during the ASCO session discussion. The presenters all agreed that testing of WBC for mutations can complement the circulating-free DNA or tumor tissue tests and address the issue of clonal hematopoiesis.

The Zehir study — published in JAMA Oncology — brings attention to the issue of clonal hematopoiesis and its potential impact on interpreting tumor tissue sequencing results and potentially liquid biopsy results from circulating-free DNA measurements. This issue is important because of available tumor sequencing tests where normal tissue is not analyzed or how popular clinical use of liquid biopsy has become for various diseases, such as lung or colorectal cancer, among others. One of the conclusions of the study is that clonal hematopoiesis can lead to erroneous reporting of mutated DNA from tumor tissue; this may also apply to liquid biopsy tests looking for circulating-free DNA.

It is important to keep in mind that liquid biopsy tests continue to evolve, and the issue may be less of erroneous reporting than erroneous interpretation when liquid biopsy tests are performed. This point is perhaps best made by considering two different patients with advanced metastatic colorectal cancer each with a KRAS mutation in their liquid biopsy. The amount of DNA that is present — referred to as allele frequency — and the context in which this mutation is found, matter a great deal. Let’s say for the KRAS mutation, patient 1 has 25% allele frequency, whereas patient 2 has 0.5% allele frequency. Now let’s say both patients have 20% to 30% allele frequency for mutated p53 and APC genes typically found in the blood of patients with advanced metastatic colorectal cancer. In patient 1, there is a lot of circulating mutated KRAS in amounts similar to the other mutated genes, whereas in patient 2 the mutated KRAS is very minor. For patient 1 with a high mutant KRAS allele frequency, there is clinical experience to date that would suggest such patients have the mutation in the tumor and should be spared from anti-EGFR therapy. For patient 2, the interpretation could be that the minor mutant KRAS allele frequency found in the liquid biopsy is from a rare subclone due to tumor heterogeneity or could be from clonal hematopoiesis. In such a case, the clinician needs to be careful with interpretation of the test result. Some expertise with cancer genetics, underlying biology of various disease mutations and allele frequencies is relevant here as to how the test results are interpreted. For patient 2, the origin of the low allele fraction may be possible to resolve by sequencing WBC and/or the patient’s tumor tissue. It is important to be clear that the result of the circulating-free DNA liquid biopsy is not necessarily erroneous, but it can be erroneously interpreted. One solution is to do WBC or normal cell sequencing on all patients; such a solution will ultimately depend on associated costs.

In our study — also presented at this year’s ASCO meeting — of liquid biopsy in a cohort of 33 patients with metastatic colorectal cancer, we made the point that context matters. When the liquid biopsy test is completed, the status of the tumor and its therapy are relevant, as is whether concomitant mutations associated with colorectal cancer are found in the circulating-free DNA. There are various scenarios in which it may not be necessary to sequence WBC, for example in the context of serial liquid biopsy looking at an estimate of tumor burden, if one is looking for actionable targets that may not have otherwise been discovered, or if the goal is to uncover drug-resistance mechanisms. If there is uncertainty about interpretation, as is often done in medicine, secondary testing can be performed without subjecting all patients to complicated testing of both circulating-free DNA and WBC upfront and each time they need a liquid biopsy.

It is important to note that in the JAMA Oncology paper, one 77-year old patient with colorectal cancer was found to have a KRAS mutation (G12R) at an allele frequency of 0.087 in WBC and 0.041 in the tumor. This was one patient out of 1,538 patients with colorectal cancer, and whether the patient’s tumor analysis showed a high allele frequency of common colorectal cancer mutations — such as APC and p53 — was not mentioned. Although it is clear in this rare patient that the mutant KRAS was detectable in both WBC and tumor tissue, how the result is interpreted by the clinician as actionable and whether such a patient would be deprived of anti-EGFR therapy is open for debate. It is not completely clear that the origin of the KRAS was not the tumor or a subclone within the tumor that is more aggressive in terms of shedding into the blood, although the fact the allele frequency was enriched in WBC argues for clonal hematopoiesis in this patient. However, to complicate things even more, it is worth remembering that there are sometimes clusters of tumor cells that may be attached to WBC or macrophages in the WBC population that may have engulfed tumor cells.

Clearly, the position of sequencing WBC has merit and may also address germline disease-associated findings, but a question is whether this is necessary for each and every patient every time they have a liquid biopsy or tumor tissue sequencing. The answer may be yes if the cost of the test comes down and is not significantly increased due to the additional testing. Given how rare the metastatic colorectal cancer patient was in the study, and the idea that some liquid biopsies are done serially for tumor monitoring, it still brings up the question as to whether WBC sequencing needs to always be done every time a tumor tissue or circulating-free DNA is sequenced.

At the ASCO session, I asked the presenters and discussant whether it could be considered malpractice to not do concomitant normal cell — WBC or buccal swab — testing along with each circulating-free DNA test. The consensus was that WBC sequencing is cheap and should be done, but it wouldn’t be malpractice not to do it as the field is still learning from the experiences. It is clear there will be increasing debate about the issue of clonal hematopoiesis and the interpretation of tumor genomic tests from tissue or blood. Clinicians need to be aware of the issue to be able to determine what to do with the information that is available from tumor tissue and/or circulating-free DNA testing on their patient. As the issues become more complex and technically involved, the need for experts with the molecular genetics and tumor biology expertise increases as does input from molecular tumor boards. The tumor testing companies can also add language to their reports to specifically address the issue of clonal hematopoiesis and whether this could be a concern for a particular patients’ molecular profiling result.

 References:

Swanton C, et al. Abstract 12003. Presented at: ASCO Annual Meeting; June 1-5, 2018; Chicago.

Zehir A, et al. Abstract 12004. Presented at: ASCO Annual Meeting; June 1-5, 2018; Chicago.