New antimyeloma drugs in spinal cord infiltration for multiple myeloma. Determination of lenalidomide in cerebrospinal fluid with ultrasensitive high-performance liquid chromatography

doi:10.4236/mc.2012.12003

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Vol.1, No.2, 11-13 (2012) Modern Chemotherapy

http://dx.doi.org/10.4236/mc.2012.12003

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: [email protected]

Received 29 August 2012; revised 30 September 2012; accepted 8 October 2012

ABSTRACT

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

Chromatography

1. INTRODUCTION

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. CASE REPORT

2.1. Clinical and Laboratory Findings at

Diagnosis

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-

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%.

3. RESULTS

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.

4. DISCUSSION

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

I. Rapado et al. / Modern Chemotherapy 1 (2012) 11-13

[2] Chang, H., Bartlett, S.E., Patterson, B., Chen, I.C. and

Long, Y.Q. (2005) The absence of CD56 on malignant

plasma cells in the cerebrospinal fluid is the hallmark of

multiple myeloma involving central nervous system. Bri-

tish Journal of Haematology, 129, 539-541.

doi:10.1111/j.1365-2141.2005.05493.x

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

nervous system myelomatosis: Review of the literature.

European Journal of Haematology, 80, 1-9.

[4] Tsenova, L., Mangaliso, B., Muller, G., Chen, Y., Freed-

man, V.H., Stirling, D., et al. (2002) Use of IMDi3, a tha-

lidomide analog, as an adjunct to therapy for experiment-

tal tuberculous meningitis. Antimicrobial Agents and Chemo-

therapy, 46, 1887-1895.

doi:10.1128/AAC.46.6.1887-1895.2002

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-

tion of the spinal cord in a patient with multiple myeloma.

Journal of Clinical Oncology, 26, 4207-4209.

[6] Dimopoulos, M.A. and Kastritis, E. The role of novel

drugs in multiple myeloma. Annals of Oncology, 19, 121-

127. doi:10.1093/annonc/mdn444

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

intrathecal liposomal cytarabine in a patient with men-

ingeal myelomatosis. British Journal of Haematology,

138, 812- 823. doi:10.1111/j.1365-2141.2007.06712.x

5. ACKNOWLEDGEMENTS

[9] Richardson, P

.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

myeloma. Blood, 100, 3063-3067.

doi:10.1182/blood-2002-03-0996

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.

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