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Chemotherapy-induced Damage to the CNS as a Precursor Cell Disease

Project Overview

An increasing number of studies indicate that adverse neurological effects of systemic treatment with chemotherapy occur with a frequency that suggests these poorly understood consequences of cancer treatment may affect more individuals than many of the most intensively studied neurological diseases. Despite the increasing recognition of the importance of understanding the adverse neurological effects of cancer treatment, little is known about the biological basis for these problems.

In research that has been referred to as a “wake-up call to neuro-oncologists ” (Duffner, 2006), we set out to investigate the biological foundations underlying the adverse effects of systemic treatment with chemotherapy on the brain. Our results indicate that precursor cells of the brain, and also myelin-forming oligodendrocytes, may be more vulnerable to chemotherapeutic agents than are cancer cells themselves, and that even transient exposure to systemic chemotherapy is sufficient to create severe delayed damage in the CNS.

As an example of the potential severity of the neurological problems associated with systemic exposure to chemotherapy, current data suggest 18% of all breast cancer patients receiving standard dose chemotherapy manifest cognitive defects on post-treatment evaluation. Moreover, such problems are reported in over 30% of patients examined two years after treatment with high-dose chemotherapy, a > 8-fold increase over the frequency of such changes in control patients. Even these numbers may be underestimates of the magnitude of this problem, as two longitudinal imaging studies on breast cancer patients treated with cocktails of high-dose chemotherapy have shown that white matter changes in the CNS could occur in up to 70% of individuals. Neurological complications observed as a consequence of systemic treatment with chemotherapy include leukoencephalopathy, seizures, cerebral infarctions, changes in electrophysiological indices of information processing, reductions in general quality of life and cognitive impairment. Such side effects are associated even with systemic treatment of low-grade tumors, do not require metastases to the CNS, and have been observed with virtually all categories of chemotherapeutic agents.

Despite the increasing recognition of the importance of understanding the adverse neurological effects of cancer treatment, the most basic questions needed to understand this problem have only recently begun to be addressed. This lack of knowledge stands in striking contrast with the many studies on the effects of irradiation on the CNS, despite the fact that whereas radiation damage is only caused by therapy targeted to the CNS, toxicity after chemotherapy also occurs after systemic administration of these compounds and does not required targeted CNS delivery. Despite the clinical importance of this problem we are still in the early stages of determining which cells are vulnerable, whether vulnerability is restricted to dividing cells, whether toxicity is due to direct or indirect effects, how the sensitivity of primary neural cells compares with that of cancer cells, what the effects are of chemotherapy on different CNS cells in vivo, whether chemotherapeutic agents with different modes of action target the same or different populations of normal cells and whether systemic treatment with chemotherapy causes syndromes of acute and/or delayed CNS damage even in the absence of ongoing disease processes (such as cancer itself).

CNS precursor cells are as or more vulnerable than cancer cells to multiple chemotherapeutic agents
In our first studies (Dietrich et al., 2006) we found that normal neural progenitor cells and oligodendrocytes of the CNS were exceptionally vulnerable to the toxic effects of the DNA cross-linking agents BCNU and cisplatin and to cytarabine, an anti-metabolite. Vulnerability to these agents was observed for all classes of lineage-restricted progenitor cells that can be readily grown as purified cell populations. Moreover, vulnerability was not restricted to dividing cells, as non-dividing oligodendrocytes were also targets of these agents. Comparative analysis of multiple cancer cell lines from different tissues showed that most such cell lines were more resistant to these agents than primary cells (despite often being chosen for their previous employment in studies on response to the chemotherapeutic agents studied). Thus, it is appears that the vulnerability of multiple normal cell populations of the CNS to cisplatin, BCNU and cytarabine rivals or exceeds the vulnerability of cancer cells themselves. Moreover, the toxicities seen in our studies occurred well within the cerebrospinal fluid concentration ranges achieved for these agents during cancer therapy. Toxicity of BCNU, cisplatin and cytarabine in vitro also was associated with suppression of cell division of O-2A progenitors even when these agents were applied transiently at exposure levels that caused little or no cell death and that represent small fractions of the cerebrospinal fluid concentrations achieved with systemic application of chemotherapy during cancer treatment.

In vitro studies accurately predicted outcomes obtained when the same agents were studied in vivo. In these experiments, animals received 3 intraperitoneal injections of a single agent spread over a 5 day period. Subsequent analysis revealed death of both neuronal and glial precursors, as well as of oligodendrocytes. We also observed prolonged reductions in cell division in vivo in the hippocampus, the subventricular zone and among O-2A progenitor cells in white matter tracts.

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CNS cells and oligodendrocytes are more sensitive to BCNU and cisplatin than many cancer cells. Cells were treated with cisplatin (a, b) and BCNU (c,d) over a wide dose range (0.1 µM-100 µM and 5 µM-200 µM, respectively). Similar results were obtained in studies on human precursor cells and other cancer cell lines. For both drugs shown here, and also for cytarabine and 5-FU {Dietrich, 2006 #4693; Han, 2008 #6962}, there were no drug concentrations at which tumor cell lines were more sensitive than the more sensitive neural progenitor cells or oligodendrocytes.


Treatment with 5-fluorouracil (5-FU) causes a syndrome of delayed CNS degeneration
One of the most puzzling facets of chemotherapy-induced damage to the CNS is the occurrence of toxicity reactions with a delayed onset. While this has been particularly well documented in pediatric populations exposed to both chemotherapy and cranial irradiation, it has also become clear that delayed toxicity reactions also occur in adults treated only with systemic chemotherapy. For example the white matter changes induced by high-dose chemotherapy for breast cancer often have a delayed onset of several months after treatment is completed.

To begin dissecting the biological foundations of delayed CNS damage, we first examined the effects on this tissue of 5-fluorouracil (5-FU), an anti-metabolite known to be associated with both acute and delayed CNS toxicities. Neurological symptoms may occur in some patients several months after adjuvant therapy with 5-FU.

As with our studies on cisplatin, cytarabine and BCNU (Dietrich et al., 2006), in vitro analysis of vulnerability to 5-FU revealed that lineage-restricted progenitor cells of the CNS and non-dividing oligodendrocytes were exceptionally vulnerable to the effects of this anti-metabolite at concentrations equal to or below clinically relevant exposure levels. In vitro analyses again predicted the acute in vivo effects of 5-FU with considerable accuracy (Han et al., 2008). 5-FU exposure transiently increased apoptosis, and suppressed proliferation for extended periods of time, in the SVZ, DG and CC. Cell-type specific analyses confirmed that the major populations affected in vivo were also progenitor cells and oligodendrocytes.

Further in vivo studies (Han et al., 2008) revealed that transient systemic exposure to 5-FU was associated with a syndrome of delayed white matter damage that had three features of particular interest:
(i) Early signs of neurological dysfunction of a type predicted by damage to myelin could be detected non-invasively by analysis of the auditory brainstem response (ABR). Such effects were not seen directly after treatment, but began to emerge after one week. Our ABR analyses appear to provide one of the first demonstration of adverse effects of chemotherapy on a functional outcome related to CNS myelination. This analysis of the velocity of impulse conduction from the cochlea to the CNS could provide a clinically useful means of studying both damage and the efficacy of putative therapies aimed at preventing or reversing this damage.

(ii) Eight weeks after treatment, examination of the corpus callosum, the major myelinated tract in the CNS, demonstrated transcriptional dysregulation in oligododendrocytes and marked myelin pathology. Many oligodendrocytes lost expression of Olig2, a transcriptional regulator critical in oligodendrocyte development, leading to the abnormal situation in which many oligodendrocytes were Olig-2 negative. These findings provide the first demonstration that chemotherapy alters the normal expression of important transcriptional regulators in oligodendrocytes. Ultrastructural analysis further demonstrated both myelinopathies and axonopathies, with extensive myelin blebbing, cytoplasmic inclusions and other structural changes indicative of a pathological process.

(iii) Damage was not repaired by endogenous precursor cells, and instead became worse with time after treatment. Although the corpus callosum contained abundant oligodendrocytes when animals were examined 8 weeks after treatment, examination at 6 months after treatment revealed a virtually complete loss of oligodendrocytes and of myelin basic protein (one of the major myeln-specific proteins). Thus, even a short-term exposure to 5-FU caused long-term and apparently irreversible damage to white matter tracts.

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Systemic 5-FU treatment causes delayed myelin degeneration in the CNS. A, B) Auditory brainstem response analysis enables early detection of defects in impulse conduction in the auditory nerve. Baseline ABR (auditory brainstem responses) hearing tests were performed on each animal 1 day before initiation of treatment with 5-FU. After treatment ended, follow-up ABR tests were conducted on each animal at various points during a time course of 56 days. ABR latencies were analyzed for each individual at each time point, and change of latency was calculated as Lt –L0 (Lt = latency values at Day 1, Day 7, Day 14, or Day 56 post treatment; L0=baseline latency values 1 day before treatment initiation). A, the change of inter-peak latency P2-P1 values. B, the change of inter-peak P3-P1 latency values. As seen, at later time points Day 14 and Day 56, both II-I and III-I inter-peak latency values of 5-FU treated animals showed average increases of >0.13 ms, while these inter-peak latency values of sham treated controls show average decreases or an increase of < 0.04ms. Changes in impulse conduction are >0.1 ms are generallu thought to be functionally significant in respect to auditory function. Data are Mean + S.E.M. Statistical significance of the difference between the means of control and treated groups was p <0.05 in A, p<0.01 in B (CI=95%; paired, one-tailed Student-t-test). C—I) Systemic 5-FU treatment causes delayed dysregulation of Olig2 expression in oligodendrocytes in the corpus callosum. Animals were treated with 5-FU 3 times over 5 days and analyzed for expression of Olig2 in the corpus callosum at various time points. There was a marked reduction in the number of Olig2+ cells at 56 days (C-E = control; F-H = 5-FU) after completion of treatment, but not at 1 or 14 days after treatment. C-H: Representative confocal micrographs showing loss of Olig2 expression in a subset of oligodendrocytes (labeled in green with the anti-CC1 antibody) in the corpus callosum of a 5-FU treated animal at Day 56 (D-I) in comparison with a sham treated animal at the same time point. The reduction in numbers of Olig2+ cells seen at Day 56 after treatment was not associated with an equivalent fall in oligodendrocyte numbers, as determined by analysis of CC1+ expression. In control animals, there is a close equivalence between Olig2 expression (C) and CC1 expression (D; merged image = E). (E) also shows three Olig2+/CC1- cells, which most likely represent O-2A progenitor cells. In contrast, in 5-FU treated animals there is a reduction in number of Olig2+ cells (F), but the corpus callosum of these animals contains many CC1+ cells (G) that do not express Olig2 (H). Scale bar = 25 μm. I, % -corrected number of Olig2+ cells in the corpus callosum at Days 1 and Day 56 post treatment of 5-FU, normalized to control values at each time point. Data represent averages from 3 animals in each group, shown as Mean + S.E.M. (* = P<0.001, one-way ANOVA) in comparison with control values at each time point. Scale bar=150μm. J, K). 5-FU treatment causes reduced cellularity in the corpus callsoum at 6 months post treatment. Representative images of haemotoxylin and eosin staining from the periventricula region of a control (J) and a 5-FU treated animal (K) 6 months post treatment. J shows the normal cellular density in CC of the control 6 months post treatment; K shows the CC from a 5-FU treated animal 6 months post treatment, the cellular density in the CC has markedly decreased; Scale bar=100μm.

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Dietrich, J., Han, R., Yang, Y., Mayer-Pröschel, M., and Noble, M. (2006). CNS progenitor cells and oligodendrocytes are targets of chemotherapeutic agents in vitro and in vivo. J. Biol. 5, e22. link

Duffner, P. K. (2006). The long term effects of chemotherapy on the central nervous system. J. Biol., 5:21. link

Han, R., Yang, Y. M., Dietrich, J., Luebke, A. E., Mayer-Proschel, M., and Noble, M. (2008). Systemic 5-fluorouracil treatment causes a syndrome of delayed myelin destruction in the CNS. J. Biol. 7, e12. link

Contact

Mark D. Noble
University of Rochester
Box 633
601 Elmwood Ave.
Rochester, NY 14642
Office: MRB 2-9625
+1-585-273-1448 mark_noble@urmc.
rochester.edu