MALDI-TOF Mass Spectrometry Analysis of Cerebrospinal Fluid Tryptic Peptide Profiles to Diagnose Leptomeningeal Metastases in Patients with Breast Cancer

Abstract

Leptomeningeal metastasis (LM) is a devastating complication that occurs in 5% of patients with breast cancer. Early diagnosis and initiation of treatment are essential to prevent neurological deterioration. However, early diagnosis of LM remains challenging because 25% of cerebrospinal fluid (CSF) samples produce false-negative results at first cytological examination. We developed a new, MS-based method to investigate the protein expression patterns present in the CSF from patients with breast cancer with and without LM. CSF samples from 106 patients with active breast cancer (54 with LM and 52 without LM) and 45 control subjects were digested with trypsin. The resulting peptides were measured by MALDI-TOF MS. Then, the mass spectra were analyzed and compared between patient groups using newly developed bioinformatics tools. A total of 895 possible peak positions was detected, and 164 of these peaks discriminated between the patient groups (Kruskal-Wallis, p < 0.01). The discriminatory masses were clustered, and a classifier was built to distinguish patients with breast cancer with and without LM. After bootstrap validation, the classifier had a maximum accuracy of 77% with a sensitivity of 79% and a specificity of 76%. Direct MALDI-TOF analysis of tryptic digests of CSF gives reproducible peptide profiles that can assist in diagnosing LM in patients with breast cancer. The same method can be used to develop diagnostic assays for other neurological disorders. One of the tumors most frequently associated with LM is breast cancer. During the course of the disease, ϳ5% of patients with metastatic breast cancer will develop symptoms caused by LM. This debilitating complication's response to therapy depends upon early treatment. However, diagnosis of LM remains challenging because 25% of samples tested are false negative at the first cytological examination of the CSF, probably because of sampling error (1).Protein expression profiling of body fluids from patients with cancer has recently become a valuable tool for obtaining information on the state of protein circuits inside tumor cells and outside the cells at the host-tumor interface (2, 3). In serum and CSF, low molecular weight proteins and peptides that are related to this altered microenvironmental "cancerous" state can be detected.We studied the differential tryptic peptide profiles in the CSF from patients with breast cancer with and without LM and in CSF from control subjects. Studying CSF has several advantages over studying serum. First, tumor cells in LM patients are located in the CSF and in the leptomeninges that are surrounded by CSF. Before their transport into serum, tumor-related proteins will therefore first be shed into the CSF. Second, the normal protein concentration of CSF is 100-to 400-fold lower than in serum (4). This results in a significant over-representation of LM-related proteins in CSF compared From the ‡Laboratory of Neuro-oncology, Department of Neurology, Dr Molewaterplein 40, 3015 GD, and § §Department

Clinical proteomics in breast cancer: a review

“…In addition, Dowling et al [83] found an increase of a 7.6 kDa bovine transferrin fragment in serum-free conditioned medium of paclitaxel resistant human breast cancer cell lines, corresponding to the increased expression of the transferrin receptor they observed in whole cell lysates. Although these results were not translated to a human in vivo setting, other studies have indeed reported an association between increased serum and CSF transferrin levels and poor clinical outcome [45,75,76]. Similarly, while ubiquitin and S100-A6 were found to decrease in lysates of human breast cancer cell lines following chemotherapy induced apoptosis [25], an aberrant expression of both proteins has also been reported in breast cancer tissue [23,73].…”

“…Lastly, cerebrospinal fluid (CSF) has also been explored for prognostic markers [75,76]. CSF is specific for the central nervous system [77], contains less total protein than serum and provides a low fluid-volume-to-organ ratio, thereby augmenting biomarker discovery [30].…”

“…As collection of CSF by invasive lumbar puncture is not applicable to healthy controls, this matrix has thus far only been investigated for prognostic purposes. In search for markers indicative of leptomeningeal metastases (LM), whole CSF samples of 106 breast cancer patients were digested with trypsin [75]. Following MALDI-TOF MS analysis of the resulting peptides, a classifier with 77% accuracy in determining LM status was constructed [75].…”

“…In search for markers indicative of leptomeningeal metastases (LM), whole CSF samples of 106 breast cancer patients were digested with trypsin [75]. Following MALDI-TOF MS analysis of the resulting peptides, a classifier with 77% accuracy in determining LM status was constructed [75]. The discriminative tryptic peptides were derived of several proteins [76].…”

Applications of genetic programming in cancer research

“…In (Dekker 2005), genetic programming is used to analyze MALDI-TOF mass spectrometry data to identify a group of protein clusters that predict leptomeningeal metastasis in breast cancer patients from spectral analysis of cerebrospinal fluid in 106 patients. 54 breast cancer patients in the cohort were diagnosed with leptomeningeal metastasis, while 52 breast cancer patients were not diagnosed with leptomeningeal metastasis.…”