Polymorphism of CAG motif of SK3 gene is associated with acute oxaliplatin neurotoxicity.

Abstract

There is no agreement on which channel is involved in oxaliplatin neurotoxicity, most investigators favouring voltage-gated sodium channels. However, the small conductance Ca(++) activated K(+) channels, encoded by the SK1-3 genes, are also involved in membrane excitability, playing a role in after-hyperpolarization at the motor nerve terminal. As the SK3 gene is characterized in Caucasians by a highly polymorphic CAG motif within the exon 1, we hypothesize that SK3 gene polymorphism may influence the development of acute nerve hyperexcitability in oxaliplatin-treated patients.
Patients eligible for an oxaliplatin-containing regimen were enrolled. Detailed neurological examination, nerve conduction studies and needle electromyography were performed before and after oxaliplatin administration. DNA was extracted by polymerase chain reaction, and each allele was isolated and sequenced.
We evaluated 40 patients. After oxaliplatin administration, 28 patients developed symptoms of neurotoxicity, which were severe in 11. Patients were divided into three groups according to neurophysiological data: G0 (normal peripheral nerve excitability [PNE]), 16 patients; G1 (mild PNE), 15 patients; G2 (severe PNE), 9 patients. Genetic analysis showed different alleles ranging from 13 to 23 CAG repeats. Patients carrying alleles containing 13-15 CAG repeats experienced a significantly higher incidence of severe nerve hyperexcitability (chi-square 48.6; df 16; P = 0.0001).
The results suggest that OXA-neurotoxicity may be related to distribution of the polymorphic CAG motif of the SK3 gene, which might modulate nerve after-hyperpolarization. The 13-14 CAG repeat allele could mark patients susceptible to acute OXA neurotoxicity.

Full text

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Polymorphism of CAG motif of SK3 gene is associated with acute oxaliplatin
neurotoxicity
Basso Michele1, Modoni Anna2, Spada Daniele1, Cassano Alessandra1, Schinzari Giovanni1, Lo
Monaco Mauro2, Quaranta Davide2, Tonali Pietro Attilio2 and Barone Carlo1.

1 Medical Oncology, Catholic University of Sacred Heart, Rome, Italy
2 Department of Neuroscience, Catholic University of Sacred Heart, Rome, Italy

Running title: SK3 and oxaliplatin neurotoxicity.
Key words: neurotoxicity, oxaliplatin, KCa channels, SK3 gene polymorphism, peripheral nerve
excitability.


Disclosures: Carlo Barone was a consultant for Amgen, Merck and Roche



Correspondence to:
Prof Carlo Barone
Medical Oncology
Department of Internal Medicine
Catholic University of Sacred Heart
Largo Agostino Gemelli n 8
00168 Rome, Italy
Tel: +390630154844
Fax: +39063017334
e-mail: carlobarone@rm.unicatt.it
peer-00626286, version 1 - 25 Sep 2011
Author manuscript, published in "Cancer Chemotherapy and Pharmacology 67, 5 (2010) 1179-1187"
DOI : 10.1007/s00280-010-1466-y

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Abstract

Purpose. There is no agreement on which channel is involved in oxaliplatin neurotoxicity, most
investigators favouring voltage-gated sodium channels. However, the small conductance Ca++ activated
K+ channels, encoded by the SK 1-3 genes, are also involved in membrane excitability, playing a role in
after-hyperpolarisation at the motor nerve terminal. As the SK3 gene is characterised in Caucasians by
a highly polymorphic CAG motif within the exon 1, we hypothesise that SK3 gene polymorphism may
influence the development of acute nerve hyperexcitability in oxaliplatin-treated patients.
Methods. Patients eligible for an oxaliplatin-containing regimen were enrolled. Detailed neurological
examination, nerve conduction studies and needle electromyography were performed before and after
oxaliplatin administration. DNA was extracted by polymerase chain reaction and each allele was
isolated and sequenced.
Results. We evaluated 40 patients. After oxaliplatin administration, 28 patients developed symptoms
of neurotoxicity, which were severe in eleven. Patients were divided into 3 groups according to
neurophysiologic data : G0 (normal peripheral nerve excitability [PNE]), 16 patients; G1 (mild PNE),
15 patients; G2 (severe PNE), 9 patients. Genetic analysis showed different alleles ranging from 13 to
23 CAG repeats. Patients carrying alleles containing 13-15 CAG repeats experienced a significantly
higher incidence of severe nerve hyperexcitability (chi-square 48.6; df 16; p = .0001).
Conclusion. The results suggest that OXA-neurotoxicity may be related to distribution of the
polymorphic CAG motif of the SK3 gene, which might modulate nerve after-hyperpolarisation. The
13-14 CAG repeat allele could mark patients susceptible to acute OXA neurotoxicity.
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Introduction

Oxaliplatin (OXA) is a third-generation derivative of platinum compounds [1]. OXA-
containing regimens are now considered standard therapeutic options for patients with metastatic
colorectal cancer [2, 3]. Overall, OXA has a good profile of toxicity but neurotoxicity is the dose-
limiting side effect. It is closely related to the 1,2 diaminocycloexane carrier ligand which enhances the
antitumour activity, but also modifies the neurological toxicity that OXA shares with other platinum
compounds [4, 5]. Cisplatin-related neurotoxicity consists of a chronic dose-dependent distal sensory
neuropathy of unknown origin, although damage to gangliar neurons in the dorsal root has been
suggested [6]. In addition, patients receiving OXA may develop acute neurological symptoms, such as
cold-induced paraesthesia and pharyngo-laryngeal dysaesthesia [7].
Clinical and electrophysiologic studies have shown that OXA gives rise to hyperexcitability at
peripheral motor nerves. In particular, in patients developing acute neurotoxicity, a single electrical
stimulus to the motor nerves produces a compound muscle action potential (CMAP) followed by
repetitive discharges. At the same time, electromyography (EMG) studies show spontaneous high
frequency discharges of motor unit multiplets [7, 8]. Overall, these findings are consistent with those
observed in neuromyotomia [9, 10], an autoimmune disease caused by auto-antibodies against voltage-
gated potassium channels (VGKC). Despite this well-known observation, the experimental evidence is
still conflicting. Indeed, electrophysiologic OXA-induced changes are not mimicked by the VGKC
blockers, 4-aminopyridine and tetraethylammonium, suggesting that OXA might not interfere with
VGKCs [11]. Other authors have shown that OXA-induced hyperexcitability may be prevented by
tetradotoxin (TTX) or carbamazepine, which in turn block voltage-gated sodium channels (VGNaCs).
Moreover, -pompilidotoxin, which slows Na+ channel inactivation, induces changes resembling those
observed in OXA neurotoxicity, whereas VGKC blockers such as apamin were not able to reproduce
OXA-induced hyperexcitability [12]. Patch-clamp technique studies performed on neurons isolated
from the central nervous system of the cockroach showed that OXA was able to alter VGNaCs through
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a pathway involving calcium ions [13]. Taken together, these data are consistent with a channelopathy
involving VGNaCs [11, 12, 13].
Following the observation that a life-threatening coma during an OXA infusion was related to severe
hypokalaemia and hypocalcaemia, we focused our attention on the role of potassium channels in OXA-
neurotoxicity [14]. SK3 (KCNN3) is a unique type of small-conductance calcium-dependent potassium
channel that is highly represented in the peripheral nervous system, but absent from the central nervous
system and muscular tissue [15]. Interestingly, a highly polymorphic CAG repeat region, essential for
the formation of tetramers, characterizes the gene encoding these channels. Among Caucasians the
length of the most common allele containing the CAG motif is about 18-19 repeats, with a range of 12-
26 [16-18]. Bearing in mind that SK3 channels have a single transmembrane domain, we assumed that
interactions between each subunit would be crucial for the efficiency of the channel. Therefore we
analyzed a group of patients treated with OXA in order to assess the influence of the genotype, i.e. the
polymorphic CAG motif of the SK3 gene, on the development of acute OXA neurotoxicity.
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Patients and methods

Patients
Patients aged between 18 and 75 years, eligible for oxaliplatin-containing chemotherapy were
enrolled in this study, provided that they had an ECOG performance status lower than 2 and a normal
neurophysiologic assessment before OXA administration. A histologically proven diagnosis of
colorectal, gastric, pancreatic or biliary cancer was required. All patients had to be OXA naïve, but
previous chemotherapy was allowed except for regimens containing cisplatin, taxanes or vinca
alkaloids. Patients with symptomatic sensory neuropathy, brain metastases, leptomeningeal
carcinomatosis or psychiatric disorders were excluded, as well as patients with history of alcohol abuse
or on vitamin supplementation. Moreover, patients treated with antiepileptic drugs, amifostine,
glutathione, glutamine, calcium and magnesium solutions and other neuromodulatory agents were not
included in the study. Patients affected by diabetes mellitus were enrolled only if neither clinical nor
neurophysiologic signs of peripheral nerve involvement were detected. Written informed consent for
both electrophysiologic studies and genetic analysis was obtained.
This study has been conducted between April 2006 and May 2007, in accordance with the rules of the
local Ethics Committee and the Declaration of Helsinki.

Endpoint of the study
The study was based on the assumption that acute OXA-neurotoxicity is related to a less
common genotype of SK3 gene. Therefore, the primary endpoint of the study was to demonstrate a
difference in the incidence of acute neurotoxicity between patients with common and patients with less
frequent genotype.



Neurological study
peer-00626286, version 1 - 25 Sep 2011