Vol.1, No.2, 11-13 (2012) Modern Chemotherapy
New antimyeloma drugs in spinal cord infiltration for
multiple myeloma. Determination of lenalidomide in
cerebrospinal fluid with ultrasensitive
high-performance liquid chromatography
Inmaculada Rapado1, Juan José Lahuerta1, Laura Montejano1, María Ángeles Montalbán1,
Laura Pares2, Joaquín Martínez-López1*
1Servicio de Bioquímica, Hospital Universitario 12 de Octubre, Madrid, Spain
2Servicio de Hematología, Hospital Universitario 12 de Octubre, Madrid, Spain; *Corresponding author: jmartinezlo@yahoo.es
Received 29 August 2012; revised 30 September 2012; accepted 8 October 2012
A 64-year-old woman with IgA kappa multiple
myeloma was treated with thalidomide-dexa-
methasone. Due to progression of the disease,
bortezomib, doxorubicin and dexamethasone
were administered, followed by autologous stem
cell transplantation. Although near-complete
remission was achieved, 5 months later, neuro-
logical symptoms appeared and the patient was
diagnosed with multiple myeloma with cells in-
filtrating the spinal cord. Bis-chloronitrosourea,
bortezomib and lenalidomide were then admin-
istered and although the patient remained neu-
rologically asymptomatic, she died 3 months
later becausee of disease progression. Lena-
lidomide entered into the cerebrospinal fluid
(confirmed by ultrasensitive high-performance
liquid chromatography), although did not im-
prove the poor prognosis of multiple myeloma
involving the central nervous system.
Keywords: Lenalidomide; Multiple Myeloma; Spinal
Cord Infiltration; High-Performance Liquid
Multiple myeloma (MM) is a malignant plasma cell
(PC) neoplasm usually restricted to the bone marrow
(BM), although extramedullary spread may occur. In-
volvement of the central nervous system (CNS) and
presence of monoclonal PCs in the cerebrospinal fluid
(CSF), occur in approximately 1% of patients [1-3]. Le-
nalidomide is a second-generation immunomodulatory
compound. There are not clinical studies designed to
determine whether lenalidomide crosses the Blood-Brain
Barrier (BBB). However, preclinical studies in rabbit and
mouse models have determined that lenalidomide crosses
the BBB [4].
2.1. Clinical and Laboratory Findings at
A 64-year-old woman was seen in October 2007 be-
cause of progressive lumbar and costal pain of 3 months
duration. She had undergone hysterectomy, treatment for
myeloma in 1996, with a mild anxious-depressive disor-
der. Laboratory tests showed moderate renal insufficiency,
anaemia, 4.2 g/dL serum M-protein (paraprotein IgA-
kappa) and immunoparesis; light chain excretion 0.8 g/L
in 24-h urine, creatinine 2.1 mg/dL, calcium 13.4 mg/dL,
2-microglobulin 6.8 mg/dL, haemoglobin 10.2 g/dL and
13% PCs in Peripheral Blood (PB). Multiple cranial oste-
olytic lesions and diffuse osteoporosis were documented.
BM aspiration showed interstitial infiltration (90% atypi-
cal PCs). Cytogenetic analysis reported t (4;14) in all in-
vestigated cells.
2.2. Clinical Course
Renal insufficiency reversed completely with dexa-
methasone, zoledronic-acid and fluids administration.
MM treatment with thalidomide-dexamethasone was
then started. MM progressed after one cycle. The treat-
ment was changed to bortezomib, doxorubicin and dex-
amethasone for 5 cycles. Near-complete remission was
achieved and the patient underwent to autologous stem
cell transplantation with melphalan 200 mg/m2 as condi-
tioning regimen. She achieved near complete remission
as maximal response after transplantation. A slowly pro-
gressive subacute numb chin syndrome (cutaneous
hypo-aesthesia limited to the region served by the mental
nerve) with weakness and paraesthesia in the upper ex-
Copyright © 2012 SciRes. OPEN ACCESS
I. Rapado et al. / Modern Chemotherapy 1 (2012) 11-13
tremities was documented after 5 months. M component
level was similar to previous studies, with negative elec-
trophoresis (positive immunofixation). Flow Cytometry
(FC) of BM aspiration showed 1.2% pathologic PC. An
extrapleural lesion of 3 cm and several new vertebral
lesions were also documented. Brain MRI was normal.
Spinal MRI showed enlarged nerve roots, diffuse spinal
arachnoiditis (L3 to L5 level), and an 8 3 cm2 mass at
D7 with enlargement of soft tissues without canal inva-
sion. Lumbar puncture revealed CSF infiltration with
800 atypical PC/L (CD56+, CD38+ and CD19– by FC).
Consequently, the patient was diagnosed with MM
with cells infiltrating the spinal cord, and treatment with
bis-chloronitrosourea (BCNU) 100 mg/m2, bortezomib
1.3 mg/m2 4, lenalidomide 15 mg per 21 days every 4
weeks and dexamethasone was started. After the first
cycle of lenalidomide, neurological signs improved:
movement, sensitiveness in the upper extremities and
numb chin syndrome resolved completely. The patient
was asymptomatic. Grade IV haematological toxicity
was observed. Only 8 PC/L were detected by FC on
lumbar puncture, with normalization of MRI findings 30
days after the first rescue treatment. An identical course
of chemotherapy was administered but systemic disease
progression was documented 20 days later. The patient
died without neurological symptoms after 3 months.
2.3. Lenalidomide Determination in Plasma
and CSF with HPLC
A high-performance liquid chromatography (HPLC)
method was developed to measure lenalidomide in PB
(serum and lithium heparin-anticoagulated plasma) and
CSF. Samples were collected 1 hour after drug admini-
stration. Specimens were obtained in the first cycle of
treatment. A CFS specimen was also collected in the
second therapy cycle. The samples were centrifuged at
1500 g for 10 min and then stored at –80˚C.
We used lenalidomide 3-(4-Amino-1-oxo-1, 3-dihydro
-2H-isoindol-2-yl) piperidine-2, 6-dione; MW 259.3;
CAS No. 191732-72-6, 99.56% purity) (Selleck Chemi-
cals LLC, TX, USA). Lenalidomide standard was dis-
solved in ethanol (1 mL) and diluted (200 nM) in the
mobile phase. Stock solutions were stored at –80˚C. An
internal standard with an appropriate retention time (RT)
was selected (Chromsystems, Instruments & Chemicals
GmbH, Munich, Germany). All used chemicals were
guaranteed reagent or HPLC grade.
A Solid-Phase (SP) extraction was done before inject
the analytes in the chromatograph, with octadecy l (C18)
cartridges (Speed SPE C18/18%, Applied Separations,
PA, USA). The steps followed for the extraction of le-
nalidomide were: 1) Cartridge conditioning with metha-
nol (1 mL); 2) SP equilibration with 0.02 M KH2PO4 (pH
7) buffer (1 mL); 3) Load of serum or plasma (300 L) or
CSF (900 L) + internal standard (100 L); 4) Wash-out
with 0.02 M KH2PO4 (pH 7) buffer (1 mL); 5) Elution
with methanol-0.02 M KH2PO4 (pH 7) (1 mL) (50:50).
Separation was performed in an isocratic chromato-
graph with a 119 UV/visible detector (Gilson, Middleton,
WI, USA) and an auto sampler (Gilson 231 XL, Gilson).
The column was C18 (Mediterranean Sea 18.5 m, 25
cm 0.46 cm; Teknokroma, Barcelona, Spain) and a
meth-anol/0.02 M KH2PO4 (pH 7) (35:65) mobile phase
at 1 mL/min flow rate were used. Wavelength was fixed
at 220 nm for lenalidomide detection. The RTs were 5.5
min for lenalidomide and 6.8 for the internal standard.
Calibration curves were constructed measuring in du-
plicate the areas of six increasing concentrations. Within-
day variation was determined from five injections at two
concentrations of lenalidomide (0.5 and 10 nM). The
limit of detection was calculated from the standard de-
viation of the areas obtained in 10 injections of the drug
at the lower concentration of the calibration curve (0.1
nM). SP extraction recovery was performed in triplicate
in plasma samples spiked with lenalidomide (7.7 nM).
The quantifiable range was 0.1 - 50 nM (R2 = 0.993).
Within-day variation coefficients were 3.3% for 0.5 nM
and 5.4% for 10 nM lenalidomide. The limit of detection
was 0.009 nM for lenalidomide concentration. Drug re-
covery from the previous extraction using SP cartridges
was 100%.
A peak in lenalidomide RT was observed in the chro-
matograms for plasma and CSF samples. However, no
peak at this RT was detected in serum samples, even with
a 1 mL serum volume in SP extraction. The lenalidomide
plasma concentration for 1 h after the first cycle of ther-
apy sample was 23.2 ± 1.0 nM. The lenalidomide con-
centration in CSF was 0.81 ± 0.03 nM for the sample
taken after the first cycle and 0.99 ± 0.03 nM for the
sample taken after the second cycle.
MM with diffuse infiltration of the spinal cord is rare,
although reports have increased recently [5]. New drugs
used for the treatment of MM [6] may contribute to this
MM manifestation due to their inability to access certain
tissues such as those in the neurological compartment
[5-7]. The special characteristics of the CNS and the
presence of the BBB could prevent these drugs from
reaching the CNS.
This aggressive relapse had a dire prognosis: survival
in this situation is 4 - 5 months [2,8]. Our patient was
treated with bortezomib, BCNU and lenalidomide. First
of them was used as a more systemic active treatment,
and the second one as an efficient treatment for tumours
Copyright © 2012 SciRes. OPEN ACCESS
I. Rapado et al. / Modern Chemotherapy 1 (2012) 11-13
Copyright © 2012 SciRes. OPEN ACCESS
[2] Chang, H., Bartlett, S.E., Patterson, B., Chen, I.C. and
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that affect the CNS due to its ability to cross the BBB
and is also used in MM. Lenalidomide was included be-
cause is an active antimyeloma drug. It is not known
whether its kinetics enables it to cross the BBB, but there
is indirect evidence that it can [4].
Lenalidomide has antimyeloma activity even at nano-
molar concentrations [9]. Lenalidomide concentration in
our patient’s CSF reached 1 nM, high enough to kill MM
cells. In addition, BCNU is active in this setting and bor-
tezomib may also enter the CSF if the BBB is breached.
[3] Nieuwenhuizen, L. and Biesma, D.H. (2008) Central
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lidomide analog, as an adjunct to therapy for experiment-
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therapy, 46, 1887-1895.
We developed and standardized a new, simple, ultra-
sensitive method based on HPLC to measure low con-
centrations of lenalidomide in plasma and other body
fluids (as low as 0.001 M). An earlier study measured
lenalidomide levels with a more complex chromato-
graphic method based on mass spectrometry [10]. Our
method is simpler and could be widely applicable in
many hospitals because it is based on widely-available
HPLC methods.
[5] V
arettoni, M., Corso, A., Zappasodi, P., Calliada, F.,
Castagnola, C., Mangiacavalli, S., et al. (2008) Infiltra-
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Although our patient’s CNS symptoms and level of
CSF infiltration improved with treatment, systemic re-
lapse occurred after the first cycle of therapy. The patient
died of disease progression but no new neurologic
symptoms appeared. New drugs used together with con-
ventional chemotherapy do not improve the poor prog-
nosis of MM with CNS involvement, even when the
drugs enter the CSF at therapeutic doses.
[7] Serefhanoglu, S., Haznedaroglu, I.C., Goker, H., Buyuka-
sik, Y. and Ozcebe, O.I. (2009) Multiple bulky cutaneous
plasmacytomas with CNS relapse without bone marrow
involvement during the course of a lambda light chain
myeloma. Onkologie, 32, 662-664.
[8] Colagrande, M., Di Ianni, M., Ciurnelli, R., Gallucci, M.,
Mariani, G. and Tabilio, A. (2007) Striking response to
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.G., Schlossman, R.L., Weller, E., Hide-
shima, T., Mitsiades, C., Davies, F., et al. (2002) Immu-
nomodulatory drug CC-5013 overcomes drug resistance
and is well tolerated in patients with relapsed multiple
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This work received medical writing support from Lidesec S.L. K.
Shashok improved the use of English in the manuscript. Lidesec S.L.
and Ms. Shashok’s fees were supported by Celgene S.L.
[10] Dahut, W.L., Aragon-Ching, J.B., Woo, S., Tohnya, T.M.,
Gulley, J.L., Arlen, P.M., et al. (2009) Phase I study of
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