Clonal hematopoiesis of indeterminate potential in patients with solid tumor malignancies

Abstract

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the expansion of cells of hematopoietic lineage that carry acquired somatic alterations associated with hematologic malignancies. The most commonly altered genes giving rise to CHIP are DNMT3A, TET2, and ASXL1. However, advanced sequencing technologies have resulted in highly sensitive detection of clonal hematopoiesis beyond these known driver genes. In practice, CHIP is commonly identified as an incidental finding in liquid and tissue biopsies of solid tumor patients. CHIP can have broad clinical consequences, given its association with hematologic malignancies and non-malignant diseases. CHIP can also interfere with next-generation DNA sequencing results, so clinicians should pay careful attention when these results are being used to guide therapy. Future research is needed to determine how solid tumor malignancies and their treatments alter the progression of CHIP, and in turn, how CHIP might be used to improve treatment selection and outcomes for patients with solid tumors.

Introduction

There are approximately one trillion blood cells arising daily from hematopoietic stem cells (HSC) in an adult human body.(1) This is sustained by an estimated 50 to 200 thousand HSC.(2) Somatic nucleotide alterations occur at approximately 1.14 mutations per cell division in cells of the hematopoietic lineage.(2) Over a lifespan, while most cells acquire somatic mutations that do not affect function, some will acquire mutations that may improve fitness and result in selective expansion of these cells without corresponding clinical manifestations.(3) This is called clonal hematopoiesis (CH). The term CH is sometimes used interchangeably with the term clonal hematopoiesis of indeterminate potential (CHIP). However, CHIP in general has a narrower definition, referring to clonal hematopoiesis in individuals without evidence of hematologic malignancies but with mutations in genes associated with hematologic malignancies, and detected at >2% variant allele frequency (VAF).(4) CHIP is associated with aging and, while it is exceedingly rare in people under the age of 40, the incidence increases with every decade of life reaching a prevalence of approximately 10 to 20% among individuals over 70 years.(5–7) However, deeper sequencing with error correction allowing for more sensitive detection of CH (i.e., smaller clones with lower VAF), does result in more frequent detection of CH in younger individuals.(8,9)

In the general population, somatic alterations in the genes associated with hematologic malignancies which gives rise to CHIP occur as the result of one of four processes known to contribute to age-associated mutagenesis: deamination of 5-methylcytosine to thymine, nucleotide insertions and deletions after double strand break repair, polymerase errors during DNA replication, and chromosomal rearrangements.(10) The mechanism of enhanced fitness likely varies based on the specific mutation and biological context.(8,11) While both driver and passenger genes can be used to detect CH, results from a number of studies suggest that most CH events, especially those detected by the more sensitive sequencing methods, may not be explained by known driver mutations. (7,12–15) Nevertheless, known cancer-associated driver genes have been more extensively evaluated in the context of CHIP. For example, mutations in DNMT3A, TET2, ASXL1, TP53, JAK2, PPM1D, ATM, CBL, SF3B1, BCORL1, GNAS, and CHEK2 are some of the most common CHIP-defining genes and constitute over 90% of all CHIP alterations (Table 1).(4,16)

Table 1.

List of 10 most common genes altered in clonal hematopoiesis of indeterminate potential (CHIP) and their function

Gene	Full Name	Function (from ncbi.nlm.nih.gov)
DNMT3A	DNA methyltransferase 3;	de novo methylation, epigenetic regulation
TET2	TET methylcytosine dioxygenase 2	demethylation, epigenetic regulation
ASXL1	ASXL transcriptional regulator 1	Chromatin binding protein
PPM1D	Protein phosphatase, Mg2+/Mn2+ dependent 1D	Suppresses p53-mediated transcription and apoptosis
TP53	Tumor protein p53	Tumor suppressor
CHEK2	Checkpoint kinase 2	DNA damage response and tumor suppressor
ATM	ATM serine/threonine kinase	Cell cycle checkpoint, DNA damage response
SF3B1	Splicing factor 3b subunit 1	Splicing machinery
JAK2	Janus kinase 2	Tyrosine kinase central to cytokine and growth factor signaling
CBL	Cbl proto-oncogene	Proto-oncogene, cell signaling

In this narrative, we summarize the prevalence and risk factors for CHIP in patients with solid tumor malignancies, review the clinical implications of CHIP among patients with cancer, and discuss ways in which CHIP might serve as a biomarker in the future for prognosis and therapeutic decision making (Figure 1).

Figure 1.

Risk factors for the development of clonal hematopoiesis and implications among individuals with solid tumor malignancies.

Risk factors

A number of risk factors for the development of CHIP have been identified. Some, but not all, demographic factors have been identified as risk factors for CHIP. Chronologic age has consistently shown to be a risk factor for CHIP, having been identified across many studies.(6,7,12,17–29) Further supporting a biologic basis associating CHIP with age, a study of 5,522 people showed that CHIP was also associated with biologic age as measured by epigenetic changes.(30) Among 19,606 women age 40–70, premature menopause was independently associated with CHIP with 36% increased odds of having CHIP, especially with DNMT3A mutations (95% 1.10–1.79; P=0.007) raising the possibility that hormonal levels, and particularly estrogen, may play a role in the development of CHIP.(31) Male sex and White race/Non-Hispanic ethnicity have also been implicated as risk factors for the development of CHIP in some, but not all studies. (16,19,20,27–29)

There are conflicting data as to whether or not there are genetic predispositions to CHIP. Two twin studies, one with 299 pairs of twins ages 73 to 94, and another with 79 twin pairs age 70–99, did not find any evidence of inherited predisposition to CHIP.(32,33) In a separate study, a germline deletion of intron 3 in the TERT gene (telomerase reverse transcriptase gene) was associated with a 37% increased risk of CHIP in a population of 262 subjects from Iceland.(12) This was replicated in a larger study of 97,631 peripheral blood whole genome sequencing study from the US-based TOPMed program.(28) Mechanistically, a reciprocal relationship between CHIP and telomerase activity was reported.(34) In addition to the association with TERT, two additional gene areas of interest were identified –an intergenic region near TET2, found among those with African ancestry, and the intergenic region between KPNA4 and TRIM59 which were associated with a 2.4 and a 1.16 fold increased risk of CHIP.(28) Further, a case control family study of patients with germline ATG2B/GSKIP-containing 14q23 duplication found that this was associated with a predisposition to develop TET2-related CHIP.(35) Additionally, a common inherited polymorphism in the TCL1A promoter was associated with slower clonal expansion. People carrying two protective alleles had up to 80% reduced odds of having driver mutations in TET2, ASXL1, SF3B1, SRSF2, and JAK2, but not DNMT3A.(36) More research is needed to better understand the possible inherited factors predisposing to CHIP.

In terms of modifiable risk factors, smoking has the most evidence of an association with CHIP (20,23,27,28,37–39), with one study showing that ASXL1-related CHIP has the greatest association with smoking (16,37). There are also some data suggesting an association between unhealthy diet (40) and vitamin C deficiency (41) predisposing to a higher prevalence of CHIP. Conversely, being normal weight, or overweight carried a lower odds (OR, 0.71 (95% CI, 0.57–0.88) and 0.83 (95% CI, 0.68–1.01), respectively) of developing CHIP compared to those who are obese (39). Inflammation may be another modifiable risk factor. For example, these hematopoietic stem cells (HSCs) may better resist inflammatory signals, leading to clonal selection in a pro-inflammatory environment.(42)

Some cancer treatments have also been linked to the development of CHIP. Cytotoxic chemotherapy has repeatedly been shown to be associated with an increased risk of CHIP. (16,20,24) Specifically, platinum chemotherapy, in particular carboplatin, (16,25,43) and topoisomerase II inhibitors (16) have been implicated in chemotherapy-associated CHIP, and result almost exclusively in TP53, PPM1D, and CHEK2 mutations (16,20,25). There was a dose response relationship between chemotherapy and CHIP suggesting that certain treatments are causally linked to CHIP.(16) Poly-ADP ribose polymerase (PARP) inhibitor therapy was also associated with an expansion of TP53-related CHIP clones among women with ovarian cancer.(43) Targeted therapies and immunotherapy with checkpoint inhibitors have not been found to be associated with the development or progression of CHIP.(16,22) Prior radiation, even short courses of radiation, have been found to increase the likelihood of developing CHIP (18,20).

Prevalence of CHIP among patients with solid tumor malignancies

CHIP is relatively common in patients with solid tumor malignancies. Four large studies have provided estimates from as low as 14% to as high as 65% prevalence among patients with solid tumors although most of these studies measure CHIP after having received treatment, which as illustrated above, could influence the presence of CHIP.(17–20) There is some evidence that certain cancers are more likely than others to be associated with CHIP. In a treatment naïve setting, patients with non-small cell lung cancer, breast cancer, pancreas, and prostate cancer have the highest prevalence of CHIP, while patients with renal cell carcinoma and ovarian cancer seem to have the lowest prevalence (16,44). Figure 2 summarizes the prevalence of CHIP across tumor types that have been estimated across studies, noting that the method for detecting CHIP varied by study. Whether or not the presence of CHIP is associated with an increased risk of the development of solid tumor malignancies is currently unknown. Certainly, given its association with the dysregulation of systemic inflammation, and the known association of inflammation with cancer, it is conceivable that it might be; however, this is currently an active area of research. (45,46) Furthermore, whether or not the mechanism of CHIP development in patients with different types of tumors is the same, and the same as the population without a cancer diagnosis, is also unknown.

Figure 2.

Prevalence of clonal hematopoiesis cited in the literature across different tumor types.

Clinical Implications for cancer risk and overall survival

CHIP has been associated with hematologic malignancies as well as non-malignant diseases. In the general population, CHIP is associated with an approximate 0.5% to 1% progression per year to an overt hematologic malignancy. (6,7,12) The malignant potential of CHIP is determined not only by the size of the clone but also by the type and number of mutated genes. The presence of mutations in spliceosome genes, IDH1/2 mutations, TP53 mutations, multiple mutations, and high variant allele frequencies are associated with an increased risk of myeloid malignancies. Interestingly, the risk appears lowest in patients with single DNMT3A or TET2 mutations. (47,48) While secondary hematologic malignancies are very rare among patients with solid tumor malignancies, the risk seems to be increased among those with CHIP. In one study, hematologic malignancies were more common among patients with CHIP (1% prevalence; 95% CI 0.5% - 1.8%) compared to solid tumor patients without a history of CHIP ( 0.3% prevalence; 95% 0.1–0.5%).(20) Case control studies found that between 62 to 71% of patients with solid tumor malignancies who did develop hematologic malignancies had antecedent CHIP compared to 27%–31% of control patients with solid tumor malignancies who did not develop hematologic malignancies.(49,50) How can we prospectively use this information? Breast cancer is one of the most common preceding malignancies to therapy-related myeloid neoplasms, an uncommon but incurable cancer with a very poor prognosis and median survival of 15 months.(51) Among women with a history of breast cancer who go on to develop therapy-related neoplasms, CHIP has been identified in tumor infiltrating leucocytes of the primary breast tumor.(52) This suggests that known pre-existing clonal hematopoiesis, especially in one of the high-risk therapy-related myeloid neoplasm genes, like TP53, may identify women at higher risk of developing hematologic malignancy following curative treatment for localized breast cancer. One study did assess if screening for CHIP can be used as a marker for the development of therapy-related myeloid neoplasms after breast cancer treatment (53). While this study did not show that CHIP was a risk factor, the median follow up time was 3.1 years, which is likely insufficient follow up to detect a difference given the median time from breast cancer treatment to therapy related myeloid neoplasm is 5–6 years. (52)

Consistently, clonal hematopoiesis is associated with lower overall survival in the general population, however, the relationship among patients with solid tumor malignancies is less clear.(6) When looking across cancers, CHIP has been associated with lower survival rates that were not driven by the development of hematologic malignancies.(20) There is a single study of metastatic colorectal cancer patients where CHIP was found to be an independent predictor of improved overall survival when considering age, treatment, extent of disease, and functional status.(24) This was most pronounced for patients with DNMT3A-mutated CHIP who also were found to have early tumor shrinkage, suggesting that there may be some relationship or interaction with certain treatments in certain cancers. (24) More research is needed to better understand if CHIP is a prognostic biomarker in patients with solid tumor malignancies, and if or how the presence of CHIP should be considered in the context of cancer clinical trials.

Clinical Implications for Nonmalignant Conditions

Outside of cancer, CHIP is associated with a number of conditions which have inflammation as a contributing etiology to the disease. The presence of CHIP is associated with 1.9 times the risk of atherosclerotic cardiovascular disease (95% CI 1.4, 2.7) and 4 times the risk of acute myocardial infarction (95% CI 2.4, 6.7) after adjusting for known cardiovascular disease risk factors.(54) This potentially has important implications for patients with solid tumor malignancies, as cardiovascular disease is a major cause of morbidity and mortality for patients with cancer.(55,56) Some therapies used for cancer treatment, for example androgen-targeting agents in prostate cancer, also increase the risk of cardiovascular disease.(57) As providers assess the risk-benefit ratio and choose certain treatments based on the side effect profile of the medications, it may be that knowing the presence of CHIP may help create a more personalized risk assessment for patients.

In addition to ischemic heart disease, CHIP is associated with stroke. In a study of 86,178 participants from 8 large cohort studies, CHIP was associated with a 14% increased risk of stroke after adjusting for age, race, and sex (HR 1.14, 95% CI 1.03 – 1.27), with a greater association with hemorrhagic stroke (HR 1.24 95% CI 1.01, 1.51) rather than ischemic stroke (HR 1.11, 0.98–1.25). This seems to vary by mutation. TET2 alterations were associated with a 90% increased risk of ischemic stroke (HR 1.18, 3.05) but no statistically significant increased risk of hemorrhagic stroke (HR 1.50, 0.87–2.61), whereas DNMT3A alterations were associated with an increased risk of hemorrhagic stroke (HR 1.44; 1.03–1.98) but no statistically significant increased risk of ischemic stroke (1.02; 0.82–1.26).(58) DMNT3A and TET2 mutations were both associated with an increased risk of hemorrhagic stroke. In the general population, CHIP has also been associated with a higher risk of heart failure,(59,60) COPD,(38,61) pulmonary hypertension,(62) autoimmune disease,(63) osteoporosis, and decreased bone mineral density.(64) Among oncology patients, CHIP was associated with an increased risk of infection – a nearly 2 times greater risk of severe COVID-19 disease, 2 times the risk of Clostridium difficile, and 56% increased risk of Streptococcus or Enterococcus infections.(65) Given the underlying associations with increased inflammation, it was hypothesized that CHIP would also contribute to the risk of VTE in oncology patients, however this has not been observed.(66)

Diagnostic Interference and misattribution

Much of the research presented above is based on the detection of CHIP for research purposes, identified by sequencing of peripheral blood mononuclear cells. In real world clinical practice, CHIP is most often inferred after being detected as an incidental finding on routine genetic testing. CHIP mutations identified on these tests can be misattributed as tumor or germline derived when in fact the mutations are somatic alterations in leukocytes.

Interference on germline genetic testing

Germline genetic testing in oncology patients is most commonly done via blood or saliva testing.(67) Results from blood samples can be contaminated by the presence of CHIP. Saliva-based samples may still contain a high proportion of white blood cells (rather than purely being buccal epithelial cells) although it varies considerably and depends on the sample acquisition technique.(68) The misattribution of germline alterations is rare, as germline alterations in many of the CHIP associated genes are associated with neurodevelopmental disorders. For example, ASXL1 germline variants are associated with Bohring-Opitz syndrome – a syndrome that presents with severe intellectual disability, developmental delay, and seizures.(69) There are some false positives where CHIP alterations are called germline genetic alterations. Among 6,060 patients undergoing germline genetic testing, pathogenic mutations were found to actually be CHIP in 18 of the patients, putting the prevalence of interference of all the patients at 0.3%, although CHIP was the most common incidental finding in this population.(70) Another study of 116,084 germline genetic tests found a prevalence of somatic interference in 0.05% of samples.(71) TP53 mutations were the most common in this cohort and besides TP53, ATM, and CHEK2, most of the genes associated with CHIP are not included on germline genetic testing panels.(71) However there are ways to distinguish between true germline alterations and CHIP interference. Among 84 patients who had germline blood or saliva-based testing where a pathogenic TP53 mutation was found, on further analysis, 11 of the 84 (13%) actually had CHIP and not true germline mutations in the TP53 gene. This was identified because patients did not meet the NCCN criteria for Li-Fraumani syndrome,(67) did not have a founder gene mutation that was identified, and subsequent testing was done, including obtaining the VAF which was much lower than would be expected for a germline alteration.(72) Therefore, among patients undergoing germline genetic testing, if testing identifies a pathogenic mutation in a cancer-associated gene but the family history does not fit with the clinical picture, or the variant allele frequency is low (i.e. < 25%), interference from CHIP could be the likely explanation.

Interference on somatic testing

Interference of CHIP in the interpretation of somatic samples is more common. The rate of blood contamination in biopsy and surgical specimens contributes to the likelihood of this, and as the tumor purity of the sample decreases, the prevalence of CHIP increases.(73) The detection of CHIP in tumor specimens is associated with an increase in lymphocytes in the tumor microenvironment but it is unclear if this is because of the association of CHIP with inflammation or if this is just due to more admixing of the sample under study.(73) This occurs in an estimate of 5% to 29% of tissue specimens sent for next-generation sequencing.(21,22,74) Among patients who are known to have CHIP, 77% of them had the CHIP variant detected in the tumor tissue.(20) Variant allele frequencies may be helpful as they should vary between that found in blood (median in one study of 4.4%) compared to the VAF in the corresponding individual’s tumor tissue (median 0.5%).(20)

Perhaps the most problematic interference occurs in the setting of liquid biopsies. Liquid biopsies are increasingly being studied and used in clinical practice. Estimates are that around 50% of mutations identified in cell free DNA actually arise from CHIP rather than tumor.(13,75) Depending on the gene alteration, this may or may not have clinical implications. In one study of cell free DNA from 69 patients with prostate cancer, 10% of the patients were found to have CHIP mutations in homologous recombination repair genes. In this study, the simultaneous use of whole-blood controls largely mitigated the problem of mistaking CHIP as tumor-derived mutations.(76) Another study of 58 patients with lung cancer however demonstrated that KRAS mutations due to CHIP may also occur but are less common (3%).(77) This is important because these results would have been considered to be tumor-derived and therefore make the patient eligible to receive certain treatments; in the first example, it would have made patients eligible to receive PARP inhibitor treatment.(76) However, there would be no anticipated benefit to the patient when taking a drug to target a somatic, presumed tumor derived, mutation when in fact the gene of interest is actually CHIP-derived. Alternatively, the identification of CHIP could also lead to misinterpretation of a cancer recurrence and potentially lead to more aggressive, but unnecessary, treatment. These scenarios highlight the ways that CHIP interference on liquid biopsies can be misleading to clinicians.

Looking ahead

There are no formal recommendations for what to do when true clonal hematopoiesis is identified for patients with solid tumor malignancies. In the setting of otherwise unexplained cytopenias, some have suggested a bone marrow biopsy to evaluate for an underlying hematologic neoplasm.(78) Regular monitoring of complete blood counts to look for evidence of clinically significant cytopenias or progression to malignant neoplasm have also been suggested.(78,79) The management of cardiovascular risk factors may also be changed based on the presence of clonal hematopoiesis.(80) Specialized multi-disciplinary clinics are also being developed including participation by cardiologists, geneticists, oncologists, counselors, social workers, and other specialized services.(81) More research is needed about the differential risk of cancer and non-cancer diseases based on the type of CHIP mutation identified. Additional research is also needed on what, if any, clinical implications there are for cancer treatment when CHIP is found. For example, in patients with TP53-associated CHIP which is associated with hematologic malignancies, the type of chemotherapy or radiation used in the treatment of a primary malignancy might be changed to an option with a lower risk of therapy-related myeloid neoplasms. Alternatively, in a man with advanced prostate cancer who is deciding on whether or not to start androgen axis targeting agents, the presence of TET2-mutated CHIP might influence treatment decisions because treatment and CHIP can be associated with cardiovascular disease. Whether or not the presence of CHIP influences clinical outcomes from cancer treatments, including from immunotherapy, is an active area of research.

The presence of CHIP can have broad clinical consequences, from interference on next-generation sequencing results, to clinical consequences. In liquid biopsies, it will be increasingly important to distinguish CHIP mutations from mutations of tumor origin by improving and standardizing the technical processes. Future research is needed to determine how solid tumor malignancies and their treatments alter the progression of CHIP, and in turn, how CHIP might be used to improve treatment selection and patient outcomes.

Summarize key findings.

• Clonal hematopoiesis of indeterminate potential (CHIP) is common among individuals with solid tumor malignancies.

• CHIP is associated with an increased risk of developing hematologic malignancies and non-hematologic diseases like cardiovascular disease, and other inflammatory conditions

• CHIP can interfere with the interpretation of next generation DNA sequencing results in patients with solid tumors