A small subset of patients exposed to paclitaxel have significant and occasionally protracted neuropathy that has a major impact on quality of life. If we could prospectively identify these patients prior to administration of paclitaxel, they might be otherwise equally well served with alternative non-paclitaxel containing regimens. Using a genome-wide association study of CALGB 40101, we have identified several genetic loci associated with onset or severity of paclitaxel-induced sensory peripheral neuropathy. One of these novel markers associated with early-onset paclitaxel-induced sensory peripheral neuropathy (FGD4, rs10771973) was replicated in both Europeans and African Americans and resides within a gene with a clearly established role in the hereditary peripheral neuropathy Charcot-Marie-Tooth disease (CMT). These findings will inform studies to test the application of genetic markers for optimization of paclitaxel selection, dosing and adverse event management. Several features of the study design and analysis support the robustness of our findings, including the prospective design, a large cohort of patients with primary breast cancer who are chemotherapy naïve and treated with single agent paclitaxel, careful collection of sensory peripheral neuropathy and covariate data, strict censoring for dose and cycle reductions for other adverse reactions and preexisting neuropathy, and the use of cumulative dose to the initial incidence of Grade 2 toxicity to account for the established effect of total drug exposure on sensory peripheral neuropathy.
The current finding that FGD4
plays a role in the development of paclitaxel-induced sensory peripheral neuropathy and/or the repair response of peripheral nerves following paclitaxel injury is consistent with the known functions of the gene. FGD4
encodes for the protein FGD1-related F-actin binding protein (Frabin) and previous studies have shown specific point mutations in FGD4
can cause the congenital peripheral neuropathy Charcot-Marie-Tooth disease (CMT4H) (21
). The disease is characterized by a slow progressive demyelination of peripheral sensory and motor neurons accompanied by distal muscle weakness and atrophy, sensory loss, hyporeflexia and skeletal deformity (25
). Paclitaxel-induced peripheral neuropathy shares some of these characteristics, including sensory loss and secondary demyelination (26
). Frabin is a guanine nucleotide exchange factor for cdc42, a Rho-GTPase that regulates cellular morphogenesis, including myelination. Several hypotheses have been proposed to explain how mutations in FGD4
might lead to demyelinating CMT4H disease, including disruption of the actin/microtubule cytoskeleton, loss of c-Jun-NH-terminal kinase (JNK) activation signals, and disruption of phosphoinositide signaling pathways, all of which could affect Schwann cell myelination and/or the bidirectional communication between Schwann cells and axons (21
The observed association between the FGD4
SNP rs10771973 and paclitaxel-induced sensory peripheral neuropathy is consistent with the hypothesis that common FGD4
polymorphisms subtly affect the development and/or maintenance of Schwann cell function. In this case, carriers of common FGD4
polymorphisms would have pre-existing subclinical abnormalities and a predisposition for toxicity. This is supported by increased risk for paclitaxel-induced sensory peripheral neuropathy in asymptomatic patients with diabetes, previous platinum drug exposures and alcohol use (3
) and early Schwann cell activation in response to paclitaxel administration (29
). Alternatively, FGD4
polymorphisms could lead to impaired repair processes such as Schwann cell remyelination and/or axonal regeneration after paclitaxel exposure. Genetic variation in FGD4
could also directly affect the response of Schwann cells to axonal injury via its ability to activate JNK (30
). A neuronal protective role for activated JNK in cultured dorsal root ganglion cells exposed to oxaliplatin has been reported (31
). Whether changes in frabin activity or expression lead to a decreased neuronal regenerative capacity and/or an increased sensitivity to paclitaxel-induced sensory peripheral neuropathy requires further study. Interestingly, FGD4
was identified through a genome-wide siRNA screen in lung cancer cell lines as a paclitaxel chemosensitizer. The chemosensitizing properties of FGD4
are related at least in part to its ability to prevent mitotic progression (32
). Whether a similar mechanism is involved in the repair response to paclitaxel-induced peripheral neuropathy is unknown.
The FGD4 rs10771973 SNP is located in the intronic region and is in tight LD with a number of other SNPs. Computational analysis of the genomic region surrounding this SNP found that rs10771972, another intronic SNP in high LD with rs10771973 in both the European and African populations, is predicted to alter conserved transcription factor binding sites for Myc-Max and USF (data not shown). One could speculate that disruption of either one or both of these transcription factor binding sites in patients carrying the rs10771973 SNP could lead to altered expression and therefore function of FGD4/Frabin.
The other two top hits from the genome-wide analysis are also of potential interest for the paclitaxel-induced sensory peripheral neuropathy phenotype. In the time to toxicity analysis, the most significant SNP was in EPHA5
, which encodes for an ephrin receptor involved in axonal guidance and regeneration following injury. Recent studies have shown that in mice EphA5 mRNA is rapidly upregulated in response to a sciatic nerve lesion (20
), and that EphA5 signaling during synaptogenesis is transduced via cdc42 (19
), the Rho-GTPase involved in Frabin signaling. A common SNP in FZD3
reached genome-wide significance in the ordinal analysis. FZD3
encodes a Wnt receptor with reported roles in neurite outgrowth (33
). In light of the biological relevance of EPHA5
and the limited size of the replication cohorts available for these studies, it will be necessary to further explore the role of these two genes in larger populations of paclitaxel-treated patients. Additional studies are also warranted for other top hits, including rs2233335 in the N-myc Downstream-Regulated Gene 1 (NDRG1
; Supplemental Table 1
). Rare mutations in NDRG1
are also associated with a different subtype of CMT (CMT4D) (34
Until the availability of genome-wide approaches for identifying genetic predictors of paclitaxel-induced peripheral neuropathy, candidate gene approaches focused mostly on drug metabolizing enzymes and transporters implicated in paclitaxel exposure. These candidate gene studies yielded no replicated associations of SNPs with paclitaxel-induced sensory peripheral neuropathy, and most were complicated by a very small number of subjects, a retrospective analysis of toxicity, and chemotherapy with multiple agents (35
). In the current analysis, no significant associations were observed for any SNPs residing in candidate genes known to influence paclitaxel exposure (Supplemental Table 3
), providing further evidence that factors contributing to the function and repair of peripheral nerves are more important than alterations in paclitaxel pharmacokinetics for determining genetic susceptibility to this toxicity. Interestingly, recent analyses of peripheral neuropathy induced by treatment with bortezomib, thalidomide and vincristine have provided evidence that genes involved in repair mechanisms, inflammation, peripheral nervous system development and mitochondrial dysfunction could influence an individual patient’s risk of developing toxicity (39
). However there was no overlap of implicated genes with the current study (Supplemental Table 3
), suggesting that the mechanisms underlying this common toxicity might be drug specific.
To assess the potential translational implications of this finding to clinical practice, we estimated the cumulative dose level triggering an event for each FGD4 rs10771973 genotype. Considering the data in , to control the probability of experiencing a neuropathy event at a critical threshold of 33%, the tolerated cumulative dose level for patients with two copies of the risk allele is 710 mg/m2. The corresponding expected critical dose level for patients with one copy of the risk allele is increased to 877 mg/m2. Patients with no copies of the risk allele are expected to tolerate >1047 mg/m2, corresponding to the full dose of paclitaxel for six cycles. If these thresholds are prospectively validated and further refined in follow-up studies, they may be used to estimate tolerable dose levels based on genotype and to tailor the treatment regimen.
While this pharmacogenetic study has several advantages over previous studies on paclitaxel pharmacogenetics, including a large cohort of treatment naïve patients receiving single agent paclitaxel and a genome-wide approach to discovery, several limitations also exist. The most significant limitation is the sole use of the NCI-CTC for assessment of sensory peripheral neuropathy. It is widely recognized that detailed patient-reported symptom data and a quality of life assessment more accurately describes this phenotype and that physician reported NCI-CTC grading underreports peripheral neuropathy (43
). However, it remains difficult to apply these techniques across the multiple sites and large sample sizes required for sufficient power for pharmacogenetic analyses. In a recent Phase III study of 1,060 women treated with taxanes, the Patient Neurotoxicity Questionnaire and the Functional Assessment of Cancer Therapy-General were administered to only the first 300 patients in the study (46
). The only use of patient-reported toxicity data and symptom measurements for pharmacogenetic analysis of taxane peripheral neuropathy is limited by the very small sample size of the study (38
). While it will be important in follow-up studies to validate these findings using additional instruments, it should be noted that despite its limitations the NCI-CTC scores are widely accepted for primary evaluation of treatment toxicity in large Phase III studies such as CALGB 40101. A second limitation of the current study is the small sample size of the replication cohorts, a common issue confronting almost all pharmacogenetic studies (47
In summary, our findings support the use of prospective pharmacogenetic analyses of well phenotyped data sets collected under controlled clinical trial settings and unbiased genome-wide genetic approaches for the identification of novel genes involved in drug efficacy and toxicity. Using a prospective design for validation and replication and a well-controlled single agent clinical study we have identified a SNP in FGD4 associated with increased risk of developing paclitaxel-induced sensory peripheral neuropathy. The involvement of FGD4 in Charcot-Marie Tooth disease, a congenital peripheral neuropathy, provides strong evidence for the biological significance of this finding. The fact that a common FGD4 SNP is associated with an increased risk of paclitaxel-induced sensory peripheral neuropathy in patients with both European and African ancestry makes it of potentially broad clinical significance. Additional SNPs in EPHA5 and FZD3 were also identified as potential risk factors for the onset and severity of sensory peripheral neuropathy. Additional samples for extension and validation of these findings are currently being collected in ongoing CALGB clinical trials of paclitaxel in the setting of metastatic breast cancer.