Stability of colon stem cell methylation after neo-adjuvant therapy in a patient with attenuated familial adenomatous polyposis
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Methylation at certain human CpG rich sequences increases with age. The mechanisms underlying such age-related changes are unclear, but methylation may accumulate slowly in a clock-like manner from birth and record lifetime numbers of stem cell divisions. Alternatively, methylation may fluctuate in response to environmental stimuli. The relative stability of methylation patterns may be inferred through serial observations of the same colon.
A 22 year-old male with attenuated familial adenomatous polyposis received neo-adjuvant chemotherapy and radiation prior to surgery for rectal adenocarcinoma. Colon crypt methylation patterns before and after neo-adjuvant therapy (62 days apart) were essentially identical with respect to percent methylation and diversity. Consistent with previous studies, methylation patterns recorded no evidence for enhanced colon crypt stem cell survival with a germline mutation (codon 215) proximal to the mutation cluster region of APC.
The inability of neo-adjuvant therapy to significantly alter crypt methylation patterns suggests stem cells are relatively protected from transient environmental changes. Age-related methylation appears to primarily reflect epigenetic errors in stem cells that slowly accumulate in a clock-like manner from birth. Therefore, life-long human stem cell histories are potentially written within and may be read from somatic cell epigenomes.
KeywordsFamilial Adenomatous Polyposis Methylation Pattern Crypt Diversity Attenuate Familial Adenomatous Polyposis Molecular Clock Hypothesis
Recent studies reveal methylation of certain CpG rich sequences increases with age in normal human colon [1, 2]. These CpG rich sequences, like most CpG islands , are unmethylated at birth and appear to somatically accumulate cytosine methylation over many years. The etiology of such age-related methylation is uncertain, but one possible mechanism is random somatic errors during DNA replication . By this mechanism, methylation would inherently increase with cell division because the CpG rich sequences are initially unmethylated. Consistent with random replication errors, methylation patterns (the 5' to 3' order of methylation) differ between cells within the same colon  and overall levels increase with age [1, 2].
Although crypt methylation patterns from multiple different aged colons are consistent with a clock-like accumulation of epigenetic errors, environmental factors may also alter methylation. Age-related methylation levels differ between individuals of the same age [1, 2, 4], which may represent life-long biological differences between individuals, stochastic differences, or transient responses to environmental changes. It may be possible to distinguish between these alternatives through serial examinations of the same colon. A somatic molecular epigenetic clock predicts the amount of additional methylation is a function of the time between examinations. In contrast, if methylation is more labile, patterns may quickly change after a severe environmental insult. Here we sample methylation 62 days apart in the same colon before and after cytotoxic neo-adjuvant chemotherapy and radiation for a rectal adenocarcinoma. Methylation patterns were essentially unchanged, suggesting crypt stem cell epigenomes are relatively protected from transient environmental changes.
A 22 year-old male presented with anemia. Colonoscopy revealed eight hyperplastic to adenomatous polyps ranging in size from 0.6 to 3.0 cm scattered through the colon, and a four cm, locally invasive, ulcerative rectal adenocarcinoma five cm from the anus. The clinical impression was attenuated familial adenomatous polyposis (AFAP), with a germline heterozygous truncating APC mutation at codon 215 (CAG to TAG; Gln to stop). The first sample for methylation analysis (Day 1) was a biopsy of normal appearing sigmoid colon.
The patient received neo-adjuvant chemotherapy consisting of three, two week cycles of oxaliplatin and 5-fluorouracil, with the last cycle ending two weeks before colectomy. The patient exhibited Grade 3 toxicity with mucositis, stomatitis, diarrhea, and parasthesia. He also received radiation therapy to the pelvis (2,500 centigrays over five days), ending 19 days before colectomy. The specimens obtained for analysis were outside of the primary radiation field.
Colectomy revealed approximately 50 scattered small hyperplastic to adenomatous polyps (0.1 to 0.3 cm), and a 0.7 cm ulcerative lesion at the site of the rectal adenocarcinoma containing a few residual neoplastic glands with superficial invasion of the muscularis propria. Lymph nodes were negative for metastatic carcinoma. The second specimen for analysis (Day 62) was taken from a normal appearing region of the sigmoid colon.
Individual crypts were isolated from the first (a colonoscopic biopsy) and last (a 1–2 cm2 patch of mucosa) specimens using an EDTA-containing solution as previously described . DNA, extracted from individual crypts placed in microfuge tubes, was bisulphite treated (to convert C's to U's, whereas methylated-C's are unchanged), and amplified by PCR at two different CpG rich regions called CSX (nine different crypts) and BGN (eight of the same crypts). PCR products were cloned into bacterial vectors and eight clones representing individual PCR products were sequenced from each crypt .
In addition to numbers of divisions since birth, methylation patterns (Figure 2) may also record stem cell dynamics. For example, crypt diversity or unique tags per crypt appears to reflect crypt stem cell numbers . This AFAP patient appeared to have normal numbers of crypt stem cells because his crypt diversity (3.22 and 2.33 average unique CSX crypt tags) was consistent with a normal colon (Figure 3). In contrast, significantly greater crypt diversity has been observed in some familial adenomatous polyposis (FAP) patients, with an average of 3.9 unique CSX tags per FAP crypt compared to 3.0 with non-FAP crypts, which translates to about 4-fold more FAP crypt stem cells . Greater numbers of crypt stem cells correlate with germline APC mutations around the mutation cluster region (MCR) that remove some but not all β-catenin binding repeats . Consistent with this observation, this patient had a truncating germline mutation (Codon 215, CAG (Gln) to TAG (stop)) that removed all β-catenin binding repeats.
The ability to extract historical information from normal appearing tissue is limited. However, recent advances in molecular phylogeny illustrate that genomes automatically record ancestry and time . Although sequences are usually faithfully copied, errors rarely occur, which are also passed from generation to generation (Figure 1). Sequence comparisons can be used to reconstruct species evolution that occurred thousands to millions of years ago. The greater the differences between sequences, the greater the likely time since they shared a common ancestor.
Sequences comparisons depend on relatively constant error rates (a molecular clock hypothesis ) because time is proportional to the number of sequence differences. Although it is possible that mutation rates vary over time from environmental changes, species histories inferred from sequences are generally consistent with relatively constant mutation rates over millions of years . In theory, molecular phylogeny can also be applied to somatic cell evolution because somatic cell genomes are also passed from cell to cell. Although somatic cell sequence comparisons are impractical because mutation rates are low relative to human lifetimes, epigenetic errors also potentially record ancestry because methylation exhibits somatic inheritance . Epigenetic replication fidelity is less than sequence fidelity because age-related methylation is readily detected in the human colon [1, 2]. Colon crypt methylation patterns have been used to infer normal human crypt niche dynamics , and that most directly adjacent adult crypts are not closely related because their methylation patterns differ . Stem cell survival can also be inferred from methylation pattern diversity (number of unique tags). For example, increased crypt diversity and enhanced crypt stem cell survival is observed in some FAP patients with truncating germline mutations that remove some but not B-catenin binding repeats . In contrast and as with this patient, germline APC mutations in the N-terminus of APC have not been associated with enhanced stem cell survival , and are associated with fewer polyps and AFAP .
A general problem with the use of sequences to reconstruct ancestry is the stochastic nature of most errors. It is difficult to prove stochastic sequence errors actually accumulate in a clock-like manner, and error rates may potentially fluctuate in response to environmental changes. Highly variable error rates would obscure ancestral information because many errors may accumulate in relatively short periods.
Although it is difficult to test a molecular clock hypothesis for species evolution, it is possible to test a somatic epigenetic molecular clock through serial examinations of the same individual. This Case Report experimentally tests the hypothesis that age-related methylation predominantly reflect stem cell errors that start to slowly accumulate from birth. Consistent with this hypothesis, methylation patterns were not significantly different after 62 days despite intervening neo-adjuvant therapy. Neo-adjuvant chemotherapy targets rapidly dividing cells and normal colon is relatively unaffected, although diarrhea (Grade 3 toxicity) may and did occur in this patient. However, the therapy did not appear to alter stem cell methylation patterns, suggesting stem cells are protected from this type of therapy. Therefore, age-related methylation appeared to be relatively stable and primarily a function of the time or number of stem cell divisions since birth. Further serial examinations with more patients and longer intervals may provide additional evidence supporting or refuting this hypothesis.
In summary, age-related methylation appears to slowly accumulate in a clock-like manner in stem cells from birth. The stochastic nature of somatic epigenetic errors requires an algorithm for interpretation, which essentially translates the sophisticated methods used to extract ancestry from species genomes to reconstruct somatic cell histories. The unseen fates of human crypt stem cells may be potentially read from anyone because methylation automatically records their autobiographies.
We thank the assistance of the patient who cooperated with this study. Written consent was obtained from the patient for publication of this study. We thank the technical assistance of Mario Campuzano. This work was supported by a grant from the National Institutes of Health (DK61140).
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