Cerebrospinal Fluid Cytology and Clinical Analysis of 34 Cases with Leptomeningeal Carcinomatosis

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1 The Journal of International Medical Research 2009; 37: Cerebrospinal Fluid Cytology and Clinical Analysis of 34 Cases with Leptomeningeal Carcinomatosis J LIU*, H JIA*, Y YANG, W DAI, X SU ANDG ZHAO Department of Neurology, Xijing Hospital of the Fourth Military Medical University, Xi an City, Shaanxi Province, China The clinical characteristics and cerebrospinal fluid (CSF) cytological features of 34 hospitalized patients with leptomeningeal carcinomatosis (LC) were studied. Most patients presented with signs of meningeal irritation (19 cases) and intra-cranial hypertension (23 cases). Computed tomography (CT) and/or magnetic resonance imaging (MRI) revealed brain parenchymal lesions, hydrocephalus and leptomeningeal enhancement (nine, six and eight cases, respectively). The CSF changes included high opening pressure (21 cases), increased white blood cell count (23 cases), elevated protein levels (25 cases) and low glucose levels (17 cases). Malignant cells were found in all CSF specimens and 32 cases had malignant cells in their initial CSF examinations. High serum levels of carcinoembryonic antigen (CEA) occurred in 11 patients. Signs of meningeal irritation and intracranial hypertension were common. It is concluded that serum CEA measurement along with CT and MRI scanning are helpful in the diagnosis of LC. Crucially, however, CSF cytology could be the most important technique for the diagnosis of LC. KEY WORDS: LEPTOMENINGEAL CARCINOMATOSIS; CEREBROSPINAL FLUID; CYTOLOGY Introduction Leptomeningeal carcinomatosis (LC), previously known as carcinomatous meningitis, is a devastating neurological complication of cancers; occurring in 3 8% of all cancer patients, it is associated with major neurological disability and high mortality. 1 LC arises from either solid tumours or haematological malignancies. Malignant cells not only disseminate throughout the subarachnoid space and *J Liu and H Jia contributed equally to this article. leptomeninges, but also invade brain parenchyma along perivascular spaces. When a tumour has spread to the leptomeninges and subarachnoid space, it gains access to all regions of the central nervous system (CNS). 1,2 Most frequently seen in widely disseminated and progressive disease, 3 LC is receiving increasing attention. It is, however, sometimes difficult to diagnose and misdiagnosis often occurs as a result of variable clinical signs and symptoms. It is widely accepted that the most important 1913

2 clinical feature of LC is multiple neuraxis syndrome. 3 6 Glass et al. 7 concluded that malignant cells in the cerebrospinal fluid (CSF) indicated the presence of a malignant tumour in the CNS. A number of studies have proven that the cytological identification of malignant cells in the CSF remains the gold standard test for LC. 3,7,8 With the development of new methods, such as computed tomography (CT), magnetic resonance imaging (MRI), the detection of tumour markers and immunohisto - chemistry, the diagnosis of LC is gradually improving. The present case series was set up to evaluate the clinical characteristics, particularly the cytological features of LC, to investigate the potential origins of malignant cells and to improve diagnosis of LC. Patients and methods PATIENTS This retrospective analysis included patients who were admitted to Xijing Hospital of the Fourth Military Medical University, Xi an City, Shaanxi Province, China, between April 1999 and February 2006 with symptoms of intra-cranial hypertension, dyspnoea or disturbance of consciousness and, following a complete medical check-up, were found to have malignant cells in their CSF. This study was approved by the Human Ethics Committee of Xijing Hospital and written informed consent to participate in the study was obtained from all the patients. ASSESSMENTS Clinical data from all patients were retrospectively studied, including: clinical presentation; radiological investigations (contrast-enhanced CT and gadoliniumenhanced MRI); electroencephalogram (EEG) scans performed in our hospital; blood tests for tumour markers (including carcino - embryonic antigen [CEA], α-fetoprotein [AFP], neuron-specific enolase [NSE], and cancer antigen [CA]125 and CA199); immunoglobulin levels in the CSF; and biochemical analysis and cytological examination of the CSF. In preparation for cytological examination, cells from the patients CSF samples were collected on the slides by centrifugation of 0.5 ml at 57 g for 3 5 min using a centrifuge invented by one of the authors (X Su). After being air-dried and fixed in ethanol, the specimens were stained by the May Grünwald Giemsa (MGG) method. 9 Light microscopy was used to examine the morphological details of the MGG stained specimens at 40 and 100 magnifications. The original sites of CSF malignant cells were ascertained by studying the histopathology, imaging and clinical presentations of the patients. Results CLINICAL PRESENTATIONS Thirty-four patients (17 women, 17 men) were included in the study. The median age of the 34 patients was 51 years (range years; mean ± SD 51.4 ± 15.8). The course of the disease varied from 3 days to > 2 years on patients admission to hospital. Many patients had intra-cranial hypertension early in the course of their disease and experienced signs/symptoms such as headache, psychological changes, nausea and vomiting (23/34 patients). The main other signs/symptoms included glosso - pharyngeal paralysis (four cases), dyspnoea with chest pain (three cases), paraplegia (two cases), myasthenia (two cases) and disturbance of consciousness (one case). Concomitant clinical presentations were: 19 cases showing signs of meningeal irritation (nuchal rigidity); eight cases of seizures; 1914

3 seven cases with psychiatric symptoms (including delirium, visual and auditory hallucination and violence); six cases of significant weight loss (mean ± SD 8.63 ± 1.25 kg in 6 months) or dyscrasia; eight cases showing signs of cranial nerve lesions (including III, VI, VII, IX, X and XII nerves); four cases of reduced hearing; three cases of decreased visual activity or acroisia; and one case of severe anaemia. There was also one case each of ataxia, unsteadiness of gait, limb pain, paraplegia and hemiplegia. NEUROIMAGING Thirty-four patients underwent cranial or spinal MRI and/or CT. Lesions in the brain parenchyma involving the parietal lobe, temporal lobe, thalamus, brain stem and subependyma were noted in nine cases, a small number of which were multiple lesions. Imaging of the brain revealed meningeal thickening or leptomeningeal enhancement in eight cases. The meninges of the basilar region displayed an extensively dispersed long T1-weighted MRI signal in one case with melanoma (Fig. 1), and six cases had hydrocephalus. Abnormal signals were also found in the pituitary gland (one case) and in the choroid plexus (one case). In two cases, the thoracic segments of the spinal cord showed abnormal signals. In 13 cases, abnormalities were not identified by the use of imaging. ELECTROENCEPHALOGRAMS The EEGs of patients with seizures (eight cases) displayed an epilepsy-form pattern, and slight or medium abnormal EEGs were noted in six cases, most of which were extensive low-power slow waves. LABORATORY EXAMINATIONS Lumbar puncture data revealed high opening pressure (200 to > 400 mmh 2 O) of the CSF in 21 cases; two cases were < 60 mmh 2 O and eight cases were normal. Lateral ventricular drainage was carried out in three cases without measuring pressure. Twenty-three cases were noted to have an increased white blood cell (WBC) count FIGURE 1: Cranial T1-weighted magnetic resonance images of a patient with melanoma showing extensive enhancement along the basilar region, cisterna ambiens and cortical sulci, implying the meningeal dissemination of melanocytes 1915

4 (range /mm 3 ; mean ± SD 35 ± 43 /mm 3 ; normal < 6 /mm 3 ), 25 cases showed raised protein levels (range g/l; mean ± SD 1.71 ± 1.74 g/l; normal range g/l), 17 cases showed low glucose levels (< 2.2 mmol/l; mean ± SD 1.24 ± 0.61 mmol/l) and eight cases showed low chloride levels (< 110 mmol/l; mean ± SD ± 2.5 mmol/l). Results for immunoglobulin in the CSF were: elevated immunoglobulin (Ig)G (13 cases), elevated IgA (11 cases) and elevated IgM (12 cases). In 14 patients serum CEA was measured and, in 11 of them, it was found to be high (range ng/ml; mean ± SD 104 ± 102 ng/ml; normal range ng/ml), whereas there were lower positive rates for other tumour markers, such as AFP, NSE, CA125 and CA199. CSF CYTOLOGY The CSF specimens of the 34 patients were all negative for ink staining, acid-fast staining and Gram staining for micro-organisms. Cytological examination in the CSF was performed on all the 34 cases. Thirty-two cases were found to have malignant cells in the initial CSF cytological examination, accounting for % (mean ± SD 22.3 ± 27.5%) of the WBC count. Two cases with normal biochemical markers and WBC count in the CSF were also found to have malignant cells. Cytology of the CSF revealed that the general morphological characteristics of the malignant cells consisted of large cell bodies with different appearances lining up in clusters, indistinct boundaries among the cells or pseudopodia extending from the cell membrane, basophilic cytoplasm, a high nuclear cytoplasmic ratio, nuclei conspicuous because of hyperchromatism, nuclear membrane thickening with a rough edge or reductus, inhomogenous or loosely clustered chromatin texture, an increased number of dense-stained nucleoli and increased mitosis rate or abnormal mitosis (Fig. 2). Malignant cells in the CSF caused by lung cancer metastasis typically showed large vacuolated cells, a high nuclear cytoplasmic ratio, well-distributed nuclei and raised numbers of nucleoli. In addition, a typical ring-like cell was noticed during cytological examination (Fig. 2A). The CSF preparation from patients without definite primary tumour origin typically showed conglomerate malignant cells with large cell bodies and nuclei, and an increased number of nucleoli. Pseudopodia also were seen to extend from the cell membrane, the scant cytoplasm was basophilic and the nuclear cytoplasmic ratio was increased (Fig. 2B). Malignant cells in the CSF that originated from liver cancer were often found to contain multiple nuclei in large cell bodies (Fig. 2C). When the tumour disseminated into the marrow extensively, a cluster of malignant cells with abnormal appearance and prominent large nuclei were noted in the CSF cytology smears (Fig. 2D). Melanoma cells in the CSF typically had a high nuclear cytoplasmic ratio and were of a large size. Several vacuoles were located on the border of the cytoplasm. Almost the entire cytoplasm and even the nucleus were filled with melanin granules (Fig. 2E). In the CSF specimen from the suprarenoma, granule-like substances, which were probably secretory granules, were observed in the cytoplasm (Fig. 2F). ORIGINS OF THE CSF MALIGNANT CELLS Histopathology, imaging and clinical presentations of the patients were used to determine the origins of the CSF malignant cells. In eight cases they were found to be 1916

5 A B 50 µm C D E F FIGURE 2: Cerebrospinal fluid cytology smears of leptomeningeal carcinomatosis in: (A) lung cancer, showing two large vacuolated malignant cells and one ring shaped cell; (B) a case with unclear primary tumour origin, showing an aggregate of malignant cells with large cell bodies and nuclei, an increased number of nuclei and nucleoli, and pseudopodia extending from the cell membrane; (C) liver cancer, showing a malignant cell with two nuclei; (D) a case with widespread marrow metastasis, showing a cluster of monstrous malignant cells; (E) melanoma, showing cells with melanin granules in the cytoplasm; and (F) suprarenoma, showing a granule-like substances in the cytoplasm (scale bar, 50 µm in each of A F) from lung cancer, in two cases from gastric cancer, one case with cranial CT reporting of subarachnoid haemorrhage was finally diagnosed with melanoma through CSF cytological examination, and one case each was from liver cancer, thymoma, lymphoma, suprarenoma, medulloblastoma, germ cell tumour and acute lymphocytic leukaemia, respectively. The origins were unclear for the remaining 16 cases. DIAGNOSIS AND TREATMENT The majority of the cases had been admitted to hospital with intra-cranial hypertension or meningitis. In eight cases, cancers in other parts of the body were found upon hospital 1917

6 admission. The remaining 26 cases were first-found cancers following the CSF examination. Five cases improved after treatment and were finally discharged from hospital. Five cases were discharged because of poor response to chemotherapy and/or radiotherapy. Three cases died during their hospital stay. Twenty-one cases gave up further treatment and were discharged when diagnosis of LC was made. Discussion In LC, malignant cells infiltrate the leptomeninges or the CSF cavity by means of direct extension from primary CNS tumours and haematogenous spread or lymphatic metastasis from solid tumours in other systemic organs Misdiagnosis of LC occurs frequently because of its diverse clinical symptoms. It is, therefore, of great importance thoroughly to recognize its presentation and to improve diagnosis. Previous studies have revealed that the clinical signs and symptoms of LC are associated with increased intra-cranial pressure, such as headache, psychological changes, or nausea and vomiting, or are related to infiltration of the nerves, resulting in local neurological deficits. 1,2,4,13 15 Middle-aged and elderly patients without gender variance accounted for the majority of the 34 cases in the present study. Obvious intra-cranial hypertension and signs of meningeal irritation were often found first. In addition, some cases with LC who showed low-grade fever, emaciation, fast erythrocyte sedimentation rate, elevated CSF opening pressure, low glucose and chloride, and increased immunoglobulin, could easily be misdiagnosed with infectious meningitis or encephalitis. Thus, more attention should be paid to the differential diagnosis of LC from other conditions. Differential diagnosis should be based on the medical history and clinical picture as well as on the CSF findings and other specific laboratory tests. The majority of cases (62% [21/34 patients]) were found to be abnormal on imaging, whereas only 24% (8/34 patients) showed thickening of the meninges or an enhancement effect. Consequently, cranial MRI and CT were effective technologies in finding abnormalities, but were non-specific. It appeared that gadolinium-enhanced MRI was superior to contrast-enhanced CT, although false-negative results of 30% for the former and 58% for the latter have been reported. 16 Moreover, only 18% (6/34 patients) of cases showed an abnormal EEG, mostly extensive low-power slow waves without specificity. Increased CSF opening pressure has been previously reported in 50 70% of patients with LC, 17 and elevated CSF protein and low glucose in approximately 75% and 40% of cases, respectively. 5,7,18 20 In the present study, 62% (21/34 patients) of cases were observed with high CSF opening pressure, and 74% (25/34 patients) and 50% (17/34 patients) had increased CSF protein and decreased glucose levels, respectively, which is consistent with the results of these former studies. All 34 patients in the present study received a final diagnosis of LC following the identification of malignant cells in the CSF, hence CSF cytology was the most useful and crucial method of diagnosing LC. Malignant cells were found in the initial CSF cytological examination in 94% (32/34 patients) of cases. With such a high sensitivity provided by cytological examination of the CSF, to ensure the validity of the diagnosis, the authors believe that it is important to perform CSF cytological re-examinations in patients where LC is clinically suspected. Some researchers also believe that, although LC can be diagnosed when malignant cells 1918

7 are detected by cytological examination of the CSF, repeat examinations might be necessary to establish the diagnosis. 3 Neoplastic cells in the CSF samples of the 34 cases with LC in the present study, especially the malignant ones, were easily recognized. They had large cell bodies and nuclei, a shifted nuclear cytoplasmic ratio, intense staining, increased quantity and size of nuclei and nucleoli, active mitosis, were lined up in clusters, and had an indistinct cell membrane margin. Nevertheless, the sites from which the malignant cells had originated were usually difficult to find. One case, in which cranial CT indicated subarachnoid haemorrhage, was finally diagnosed with melanoma through CSF cytological examination; the cytoplasm of the melanoma cell was full of melanin granules, which were easily identified. Serum CEA with a high positive rate in 79% (11/14 patients) of cases was a convenient screening marker, which helps when deciding whether CSF CEA should be tested. Previous studies have measured the tumour markers, CEA and epithelial membrane antigen, in CSF by enzyme immunoassay and immunocytochemistry, respectively. 21,22 Several years ago, a case of LC from oesophageal basaloid carcinoma was reported to have been diagnosed by quantitative reverse transcription polymerase chain reaction for CEA. 23 Such effective techniques should be advocated for improving diagnosis and could be helpful to identify the definite origin of the tumour. It has been reported that the most common malignancies associated with LC are breast cancer, lung cancer, melanoma, lymphomas and leukaemias. 2,13 Lung cancer accounts for 14 29% of all cancer patients, 9 25% of whom have LC. 18,24,25 Lung cancer was most frequently associated with LC in the present study, followed by tumours of the gastrointestinal tract. In conclusion, cranial hypertension and signs of meningeal irritation were the first and main clinical manifestations of LC; cranial nerves and spinal nerve roots were usually involved. Patients with malignant cells in the CSF showed EEG abnormalities; there was, however, no specificity. Neuroimaging techniques were of great value in detecting abnormalities but were non-specific. Measurement of serum CEA was a convenient screening marker for patients with suspected cancer and may be the most important procedure in the diagnosis of LC. Cytological examination of the CSF played a decisive role in the diagnosis of LC, but the origins of malignant cells were morphologically indistinguishable in most cases. Greater use of technologies, such as immunocytological methods, electron microscope examination, or even examinations beyond the CNS, should be used simultaneously to localize and identify tumour origins. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 30 May 2009 Accepted subject to revision 4 June 2009 Revised accepted 13 October 2009 Copyright 2009 Field House Publishing LLP References 1 DeAngelis LM: Current diagnosis and treatment of leptomeningeal metastasis. J Neurooncol 1998; 38: Grossman SA, Krabak MJ: Leptomeningeal carcinomatosis. Cancer Treat Rev 1999; 25: Wasserstrom WR, Glass JP, Posner JB: Diagnosis 1919

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