Short telomeres result in chromosomal instability in hematopoietic cells and precede malignant evolution in human aplastic anemia

Abstract

In cell and animal models, telomere erosion promotes chromosomal instability via breakage-fusion-bridge cycles, contributing to the early stages of tumorigenesis. However, evidence involving short telomeres in cancer development in humans is scarce, epidemiological and indirect. Here we directly implicate telomere shortening as a critical molecular event for malignant evolution in aplastic anemia (AA). Patients' telomere lengths at diagnosis of AA, while comparable to age-matched controls, inversely correlated with the probability of developing a cytogenetically abnormal clone. A significantly increased number of telomere signal-free chromosomal ends and chromosomal numerical and structural abnormalities were observed in bone marrow cells of patients with shorter telomeres in comparison with patients with longer telomeres and healthy subjects. The proportion of monosomy-7 cells in the bone marrow at diagnosis of AA inversely correlated with telomere length, years before the emergence of an autonomous and clinically detectable abnormal clone. Marrow cells of clinically healthy individuals carrying loss-of-function telomerase mutations and with extremely short telomeres also showed chromosomal instability in vitro. These results provide the first clinical direct evidence in humans that short telomeres in hematopoietic cells are dysfunctional, mediate chromosomal instability and predispose to malignant transformation in a human disease.

Figure 1. Short average leukocyte telomere length associates with dysfunctional telomeres and chromosomal instability

(A) Telomere length was measured pre-treatment using qPCR in 183 consecutive patients with acquired aplastic anemia (blue circles) and 175 healthy volunteers (yellow circles) and represents the y-axis, measured in T/S ratio (x-axis represent the age in years). Telomere length distribution with age was not different between patients and controls (ANOVA-F test). Solid line corresponds to the regression line for aplastic anemia patients, whereas the dashed line represents the regression line for healthy subjects. (B) Most patients with acquired aplastic anemia who eventually developed a cytogenetic abnormality had pre-treatment telomere lengths below the mean for age-matched controls. Patients who eventually developed either monosomy 7 or complex karyotype are depicted as red squares and patients who developed other karyotypic changes are represented as yellow diamonds. (C) Probability of evolution to myelodysplastic syndrome and/or acute leukemia in patients with acquired aplastic anemia and treated with immunosuppression, according to pre-treatment age-adjusted telomere length. Patients were divided into quartiles based on age-adjusted telomere length (first quartile, shorter telomeres; and fourth quartile, longer telomeres). Patients in the shorter quartile (first) had a higher chance to evolve to myelodysplasia and/or leukemia (24.5%) at 5 years in comparison to patients in the second (10.7%), third (4.8%), and fourth (9.7%) quartiles (log-rank, P=0.06). (D) Probability of evolution to monosomy 7 or complex karyotype in patients with acquired aplastic anemia treated with immunosuppression, according to pre-treatment age-adjusted telomere length. The risk of evolution to these poor prognosis karyotypes was similar (2.5%) in the fourth and third quartiles, somehow higher (8.3%) in the second quartile, and significantly higher (18.8%) in patients in the shorter telomere (first) quartile (log-rank, P=0.018).

Figure 2. Bone marrow cells with shorter telomeres show chromosomal instability

(A) Bone marrow samples collected pre-treatment were assayed for chromosome 7 aneuploidy by FISH (n=39). The percentage of monosomy 7 cells in marrow samples inversely correlated with the leukocyte telomere length (P<0.001). (B) Bone marrow samples six months after initial therapy were examined for chromosomal telomere-free ends by FISH of healthy subjects (n=5) and patients with shorter (n=12) and longer (n=7) telomeres. The percentage of telomere signal-free chromosomal ends was significantly higher in cells of patients with average length shorter telomeres than in controls or patients with average length longer telomeres (P<0.0001). (C–H) Bone marrow samples of patients with shorter and longer telomeres were analyzed for chromosomal instability by spectral karyotyping (SKY). An increased number of numeric or structural abnormalities were observed in chromosomes of marrow cells of patients with shorter telomeres. Arrows identify the chromosomal abnormalities.