Tracing ancestry with methylation patterns: most crypts appear distantly related in normal adult human colon

BACKGROUND: The ability to discern ancestral relationships between individual human colon crypts is limited. Widely separated crypts likely trace their common ancestors to a time around birth, but closely spaced adult crypts may share more recent common ancestors if they frequently divide by fission to form clonal patches. Alternatively, adult crypts may be long-lived structures that infrequently divide or die. METHODS: Methylation patterns (the 5' to 3' order of methylation) at CpG sites that exhibit random changes with aging were measured from isolated crypts by bisulfite genomic sequencing. This epigenetic drift may be used to infer ancestry because recently related crypts should have similar methylation patterns. RESULTS: Methylation patterns were different between widely separated ("unrelated") crypts greater than 15 cm apart. Evidence for a more recent relationship between directly adjacent or branched crypts could not be found because their methylation pattern distances were not significantly different than widely separated crypt pairs. Methylation patterns are essentially equally different between two adult human crypts regardless of their relative locations. CONCLUSIONS: Methylation patterns appear to record somatic cell trees. Starting from a single cell at conception, sequences replicate and may drift apart. Most adult human colon crypts appear to be long-lived structures that become mosaic with respect to methylation during aging

Phase I/II study of oral etoposide plus GM-CSF as second-line chemotherapy in platinum-pretreated patients with advanced ovarian cancer

The aim of this phase I/II study was to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities of chronic oral etoposide given on days 1–10 followed by rescue with subcutaneous (s.c.) granulocyte-macrophage colony-stimulating factor (GM-CSF) on days 12–19 as second-line chemotherapy in platinum-pretreated patients (pts) with advanced ovarian carcinoma. Cohorts of three to six pts were treated with doses of oral etoposide from 750 mg m−2 cycle−1 escalated to 1250 mg m−2 cycle−1 over 10 days, every 3 weeks. Subcutanous GM-CSF, 400 μg once daily, days 12–19, was added if dose-limiting granulocytopenia was encountered. In total, 18 pts with a median Karnofsky index of 80% (range, 70–100%) and a median time elapsed since the last platinum dose of 10 months (range, 1–24 months), 30% of whom showed visceral metastases, were treated at four dose levels (DLs) of oral etoposide on days 1–10 of each cycle as follows: DL 1, 750 mg m−2 cycle−1, without GM-CSF, three pts; DL 2, 1000 mg m−2 cycle−1, without GM-CSF, three pts; DL 3, 1000 mg m−2 cycle−1, with GM-CSF, six pts; and DL 4, 1250 mg m−2 cycle−1, with GM-CSF, six pts. All pts were assessable for toxicity and 16 pts for response. Dose-limiting toxicity (DLT) was reached at DL 4 by three of six pts, showing World Health Organization (WHO) toxicity grade 4. One patient died from gram-negative sepsis associated with granulocytopenia grade 4. Two more pts developed uncomplicated granulocytopenia grade 4. Thus, we recommend that DL 3 can be used for further phase II evaluation (i.e. oral etoposide 1000 mg m−2 cycle−1, days 1–10, followed by s.c. GM-CSF 400 μg, days 12–19). The clinical complete or partial responses in each patient cohort were: DL 1, one of three pts; DL 2, one of three pts; DL 3, three of five pts; and DL 4, two of five pts. In conclusion, in this phase I/II study, we defined the MTD and the dose recommended for the therapy with oral etoposide given over 10 days followed by s.c. GM-CSF in platinum-pretreated patients with advanced ovarian cancer. Our data demonstrate encouraging activity of this regimen and strongly support its further investigation in a phase II study