Clinica Chimica Acta, 212 (1992) 123-131 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/$05.00
123
CCA 05438
Serum neurone-specific enolase levels in patients with neuroendocrine and carcinoid tumours Rodat T. Cunninghama, Colin F. Johnstona, G. Brent Irvineb and Keith D. Buchanana aDepartment of Medicine and bDivision of Biochemistry, The Queen> University of Belfast, Northern Ireland (UK)
(Received 16th March 1992; revision received 29 September 1992; accepted 12th October 1992)
Key wora!s: Neurone-specific enolase; Neuroendocrine
tumours; Carcinoid tumours
We have examined concentrations of neurone-specific enolase (NSE) in sera from 18 patients with various neuroendocrine tumours, 26 patients with carcinoid tumours, 21 patients with non-neuroendocrine tumours and 37 control individuals. No statistically significant difference between the concentrations in patients with neuroendocrine tumours and patients with carcinoid tumours was found. However the NSE concentrations in patients with carcinoid and neuroendocrine tumours, when these two groups were combined, were significantly different from the patients with non-neuroendocrine tumours or the control individuals (P I 0.01). 38.5% of the patients with carcinoid tumours had raised NSE concentrations in serum; 55.5% of those with non carcinoid neuroendocrine tumours had raised concentrations. There appeared to be no correlation between the NSE concentrations and the extent of metastases.
Introduction Neurone-specific enolase (NSE, EC 4.2.1 .l 1) is an isozyme of the glycolytic enzyme, enolase. The enzyme occurs predominantly in neurones but has also been located in neuroendocrine cells [ 11. Correspondence to: Dr. R.T. Cunningham, Department of Medicine, Wellcome Research Laboratories, Grosvenor Road, Belfast, BT12 6BJ, UK.
124
Neoplasms of neuroendocrine cells are often associated with an overproduction of a hormone which in turn gives rise to a clinical syndrome. Zollinger Ellison Syndrome, for example is associated with overproduction of gastrin, while carcinoid syndrome is associated with 5hydroxytryptamine and tachykinin release. Some neuroendocrine tumours, however, may be clinically ‘silent’, possibly because a peptide is not secreted, or is abnormal, or not associated with a syndrome. Alternatively, the target cell receptors for that hormone may be abnormal, or an inhibitor may be co-secreted. The proposal that NSE would act as a universal marker for all neuroendocrine tumours including ‘silent’ tumours has resulted in immunocytochemical (ICC) studies in small cell lung cancer [2], tumours of the gastrointestinal tract [3-61, neuroblastomas [7], melanomas [8,9], pituitary adenomas [lo], etc. However non specific results have led to some debate about the reliability of ICC staining for NSE [11,1.2]. It is unclear whether these results are a consequence of using non-specific polyclonal antibodies [ 111. In addition to ICC studies there have been several assays developed to measure NSE concentrations in serum and plasma [13-281. This paper documents the results obtained when a radioimmunoassay (RIA) was used to measure NSE concentrations in sera from patients with neuroendocrine tumours. Patients and Methods Blood samples were collected from 18 patients with histologically confirmed noncarcinoid neuroendocrine tumours and from 26 patients with carcinoid tumours (Tables I and II, respectively). A carcinoid tumour was identified as being a tumour of neuroendocrine origin which was also chromogranin A and serotonin positive. The ages of the groups were 52.1 i 14.1 and 57.5 f 10.2 y, respectively and the groups were 10 males, 8 females and 16 males and 10 females, respectively. Twentytwo of the patients with carcinoid tumours had metastases and 10 of the patients with other non-carcinoid neuroendocrine tumours also had metastases. Of those patients with carcinoid tumours, 4 had suffered from carcinoid crisis. Blood samples were also collected from 21 patients (Table III) with tumours which were not of the neuroendocrine system, also confirmed by a lack of staining for serotonin and chromogranin A. The ages of these patients were 62.7 f 12.3 y and the group was 3 males and 18 females. A group of 37 control individuals (19 female and 18 male) was studied. They had cardiac or respiratory disease and were free from neuroendocrine or neurological disorders. None of these patients had any known malignant disease. The mean age in this group was 50.3 f 16.0 y. Blood samples were obtained from fasted individuals, 10 ml being withdrawn from the antecubital vein. Samples were then centrifuged at 500 x g for 20 min at 4°C and the serum was divided into 0.5-ml portions before being stored at -20°C prior to assay. Measurement of serum NSE
The assay for NSE was as previously reported [ 131.NSE was purified from human brain obtained less than 12 h post mortem and specific antibodies raised to the pro-
125
TABLE I Serum NSE levels in patients with neuroendocrine tumours No Sex
Age
Nature of tumour
Tumour burden
Primary site
Sites of metastases
1 M 2F
66 66
Gastrinoma MEA type I
I+ I+
None None
23 16
3
M
39
Gastrinoma
2+
Pancreas Pancreas/ parathyroid Pancreas
I3
4
F
72
2+++
Pancreas
5 6 7 8 9
M M M F M
51 67 55 51 67
2+ I 2+++ 0 2+++
10
M
29
Gastrinomal insulinoma/ VlPoma VIPoma lnsulinoma Unknown MCT Gastrinoma MCT Unknown
Lymph nodes Liver
Pancreas Pancreas Pancreas Thyroid Thyroid/ pancreas Pancreas
II
F
53
2++
12 13
F M
46 65
VlPomal WDHA Gastrinoma Gastrinoma
14 15 I6
55 35 23 44
I8
M
54
Gastrinoma Gastrinoma Unknown Glucagonomti gdstrinoma Unknown
0
11
F F M F
I I+ 2+++
Pancreas/ liver Unknown Pancreas/ liver Pancreas Unknown Unknown Pancreas
2++
Pancreas
I 2+++
Liver None Liver None Liver Liver/ lymph nodes Liver
NSE Pgll
II
16 13 21 28 II3 24
28
None Liver
5 3
None None None Liver
1
5 6 63
Liver
25
MEA, multiple endocrine adenoma; VIP, vasoactive intestinal peptide; MCT, medullary carcinoma of the thyroid.
tein. The protein was also radioactively labelled and used in the development and validation of a RIA to NSE. Separation of bound and free protein was by the second antibody separation technique. The assay sensitivity was 1 pgll. Intra assay variation was 11.0% at 46.6 i 1.7 &l ; 6.7% at 28.8 f 1.9 &l and 7.4% at 19.7 f 1.4 pg/l. Interassayvariationwas 13.9%at81 f 11.3&l;8.2%at 10.2 f 0.8&land 13.5% at 9.5 f 1.2 &l. Each value obtained was the mean of at least 6 duplicates. Statistical analysis
Serum NSE concentrations were compared between the patients with carcinoids and other non-carcinoid neuroendocrine tumours using the Mann-Whitney U-test. The same test was also used to compare NSE concentrations in those patients with neuroendocrine tumours (including those with carcinoids), in control individuals and in patients with non-neuroendocrine tumours.
126 TABLE II Serum NSE levels in patients with carcinoid tumours No. Sex
Age
Tumour burden
Carcinoid crisis
Primary site
Metastases (sites)
NSE ati
Liver Liver/ omentum Liver Liver Liver
18 30
1 2
M M
56 64
2 +++ 2 +++
N N
Ileum Unknown
3 4 5
M M F
60 45 62
2 +++ 2 +++ 2 +++
N Y N
6 7 8 9 10 11 12 13 14 15 16 17 18
M F M F M M F M F F M F M
59 59 58 19 59 72 71 44 49 74 56 58 58
2 +++ 2 +++ 0 2+ 2 +++ 2+ 2 ++ 0 0 2+ 2 ++ 2 +++ 2 +++
N N N N
Y
Lung Lung Lung/ pancreas Ileum Ileum Ileum Ileum Ileum Ileum Unknown Caecum Appendix Appendix Lung Ileum Lungliver
19 20
M F
33 52
2 ++ 2 ++
Y N
Unknown Ileum
21
M
59
2 +++
N
22 23 24 25
M F M M
46 44 55 60
2 +++
N N
2 +++ 2 ++
Y
26
F
62
Ileum/ pituitary/ liver Lung Lung Unknown Ileum/ liver Ileum
N N N N N N N N
N N
Liver Liver Liver Liver Liver Liver Liver None None Liver Liver Liver Liver/ hilar node/ bone marrow Liver Liver/ lymph nodes Liver
8 343 13 13 16 32 7 10 10 9 7 21 7 10 19 10
34 14 8
Liver None Liver Liver
570 17 11 5
None
8
Rt%UItS
Control individuals
Concentrations of 9.1 f 2.5 &l (mean f S.D.) were recorded for our 37 control individuals. This results in an upper limit of the reference of 14.1 pgA(9.1 f 2 S.D.). In the patients with non-neuroendocrine tumours, the concentration was 9.4 f 2.9 g/l. Two patients had NSE concentrations just above the upper limit of the reference interval (16.2 and 15.6 &l).
127 TABLE III Serum NSE levels in patients with non-neuroendocrine
tumours
Sex
Age
Tumour type
Primary Site
Site of metastases
NSE Wll
M F M M F
64 77 46 75 47
Lung Colon Colon Colon Breast
Liver Liver Liver Liver None
8.7 13.0 8.5 16.2 7.2
F
56
Neck
None
9.2
F F F F F F F F
93 56 69 64 80 60 44 54
Vagina Breast Cervix Oral cavity Nose Endometrium Cervix Neck
None Bone None None None None None None
6.3 10.8 10.8 6.8 15.6 7.4 10.2 8.1
F F
60 66
Breast Unknown
Bone Bone
8.4 7.2
F
63
Breast
Bone
9.3
F F F F
71 68 50 54
Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Infiltrating duct cell Non-Hodgkins lymphoma Squamous cell Squamous cell Squamous cell Squamous cell Squamous cell Adenocarcinoma Squamous cell Non-Hodgkins lymphoma Adenocarcinoma Clear cell carcinoma Invasive lobular cell Squamous cell Squamous cell Squamous cell Adenocarcinoma
Cervix Cervix Cervix Breast
None None None Bone
6.4 10.5 5.4 11.6
Neuroendocrine tumour-bearing patients
Serum NSE levels were above the upper limit of normal in 38.5% of subjects studied with carcinoid tumours. Serum NSE values within these carcinoid patients varied from 5 to 570 pg/l. Twenty-two of the patients with carcinoid tumours had liver metastases but only 8 of these had a serum NSE above normal. Only 4 of the carcinoid patients had suffered from carcinoid crisis and two of these had a normal serum NSE value at the time of assay. Of the remaining 22 carcinoid patients, 14 had a normal NSE serum concentration. In patients with non-carcinoid neuroendocrine tumours, NSE was elevated in 55.5% of the cases studied. Ten of these patients had secondary tumours on either the liver or lymph nodes. Of the 10 patients with metastases, 7 had elevated serum NSE concentrations. Of the 8 patients with no metastases, 5 had normal serum NSE values.
128
Statistical analysis, using the Mann-Whitney U-test, revealed no significance when NSE levels in patients with carcinoid tumours were compared with those levels in patients with non-carcinoid neuroendocrine tumours. However, when both of these two groups were combined and compared with levels obtained in patients with nonneuroendocrine tumours, a significant difference was recorded (P = 0.01). Levels in both groups combined were also significantly different from those obtained from normal individuals (P = 0.001). In 4 patients who had suffered carcinoid crisis, only two had elevated serum NSE. Metastases in carcinoid patients did not correlate with serum NSE value; 2 of 4 patients with no metastases were associated with high serum NSE concentrations, while other patients with metastases had levels within the normal range. Similarly serum NSE concentration in each of these patients showed no correlation with degree of tumour burden. Neither did tumour burden correlate with serum NSE concentration in patients with non-carcinoid neuroendocrine tumours (Table I) and some of these patients who had metastases had normal serum NSE concentrations. Discussion It was observed that 38.5% of patients with carcinoid tumours and 55.5% of patients with other neuroendocrine tumours had elevated serum NSE concentrations. In total 45.5% of all patients with a neuroendocrine tumour at diagnosis had an elevated serum NSE concentration. Previous studies have looked at serum NSE in a range of neuroendocrine tumours. In patients with small cell cancer of the lung at diagnosis, elevated serum NSE has been recorded in 69%, 71.4%, 65.1%, 68%, 72%, 73%, 60% and 76.5% of all patients studied [14-211. Elevated serum NSE concentrations have also been noted in patients with neuroblastoma [7,22] and in patients with phaeochromocytoma [23]. Prinz et al. [24] studied patients with pancreatic islet cell cancers and intestinal carcinoid tumours and found elevated serum NSE in 46% of all patients studied. This compares favourably with our value of 45.5% of all patients studied with neuroendocrine tumours. Marangos and Prinz also reported that elevated serum NSE concentrations were less likely in patients with intestinal carcinoids than in patients with other neuroendocrine tumours, even if liver metastases were present. They found NSE to be elevated in 1 of 5 patients with intestinal carcinoid, while we found that 4 of 12 patients known to have ideal carcinoids had elevated serum NSE. Prinz and Marangos [25] found that 9 of 21, or 42.9% of the APUDoma patients they studied, had clearly elevated NSE concentrations and that clear elevations were only observed in patients with extensive disease. Close correlation of serum NSE concentrations and disease extent has been noted by others [14,16,17,23] and it has been suggested as a useful prognostic indicator of disease progress in patients with a range of neuroendocrine tumours [19,20]. In neuroblastomas it has been reported that 79% of patients with a serum NSE concentration less than 100 ng/rnl survived 2 years after diagnosis [22]. In addition it was noted that serum NSE concentration reflected the stage of disease with patients in stage I having considerably lower concentrations than those in stage IV. It was noted that serum NSE concentrations in patients with stage IVs disease, which is prone to spontaneous regression and therefore clinically more benign had lower
129
concentrations than those in stage IV despite heavy tumour burden. Our results did not indicate a correlation between serum NSE concentration and disease extent since some of the patients studied had low concentrations of serum NSE, despite heavy tumour burden. However Prinz and Marangos [25] also recorded normal concentrations of serum NSE in patients with neuroendocrine tumours and metastases. This may be because NSE is only released as a result of cell lysis and that in these patients, the tumour cells are healthy. All the blood samples collected from the patients studied here were collected prior to cytotoxic chemotherapy, although some patients (4, 5, 7, 11, 17 from Table I and 1-6, 15-21, 24 and 25 from Table II) were taking the long-acting somatostatin analogue, octapeptide SMS 201-995 (Sandoz AG, Basel, Switzerland). This drug relieves the symptoms of carcinoid syndrome and functioning neuroendocrine tumours, presumably by inhibiting peptide synthesis or release. Of these 20 patients on SMS 201-995, only 10 had serum NSE concentrations above the upper limit of normal. We have found previously that the circulating concentrations of NSE are dramatically elevated after intra hepatic arterial streptozotocin and floxuridine [ 131. It may be possible for healthy tumour cells to be associated with normal serum NSE concentrations, although necrotic areas are more likely in larger tumours. Two carcinoid patients studied here who had suffered carcinoid crisis had greatly elevated concentrations of serum NSE, although two similar patients had normal serum NSE concentrations. Patients 4, 19 and 25 had blood samples collected 15, 3 and 28 days before carcinoid crisis and serum NSE concentrations of 343, 10 and 11 pg/l, respectively. Blood was collected 6 days after crisis in patient 20 who had a serum NSE of 34 &l. It may be that crisis is associated with the onset of the release of the contents of the neuroendocrine tumour resulting in subsequent flushing, lacrimation, diarrhoea, etc. If this release of contents involved cell breakdown then NSE should also be released. However if NSE has a serum half-life of 48 h as reported by Ishiguro et al. [26], this would imply the need to obtain a blood sample from the patient within this period. This might account for the normal serum NSE values in two of the patients who had previously had carcinoid crisis. Alternatively, the tumour cells may release their contents but remain viable. There have been reports concerning the non-specificity of NSE with elevated serum concentrations reported in patients with tumours which are not of the neuroendocrine system [ 11,121. We found that concentrations were very slightly elevated in 2 of 21 patients with non-neuroendocrine tumours but were still less than the mean normal value f 3 S.D. Ishiguro [7] has suggested that the elevated serum NSE concentrations he has reported in 2 of 6 patients with Wilm’s tumour may be as a result of the tendency for the undifferentiated small cells present to develop into a particular cell type, e.g. smooth or striated muscle. Carney and Teeling [ 1l] have suggested that serum NSE is neither sensitive or specific for small cell lung cancer although they do suggest that it may be of use in monitoring disease progress after chemotherapy and in distinguishing paediatric tumours. They also question whether the non-specific staining obtained in immunocytochemical studies of neuroendocrine tumours may be as a result of technical problems or problems with the antibody. Thomas et al. [27] have reported a much more specific monoclonal antibody to NSE with only 2% of non-neuronal, non-neuroendocrine tumours staining positively with respect to 20% non-specific staining using a polyclonal antibody.
130
Other factors which may account for nonspecific staining in non-neuronal, nonneuroendocrine tumours includes incorrect classification of the tumour type [28], altered metabolism [29] or aberrant gene expression [ 121. There have also been reports of transformation of one cell type to another [12,30]. Whatever the reasons for non-specific staining with NSE, it remains that immunhistochemically positive results should be treated with caution. This does not however preclude studies into the viability of NSE as a serum marker especially in monitoring chemotherapy [13,17,19,25]. In conclusion, our results show that serum NSE concentrations may be elevated in patients with neuroendocrine tumours, including carcinoid tumours but that they are not always raised. Although serum NSE may not be a particularly sensitive marker for the diagnosis of neuroendocrine tumours, despite reports to the contrary, it appears to be specific. Acknowledgements This work was supported in part by the Medical Research Council, UK and by the Northern Ireland Chest, Heart and Stroke Association. References 1 2
3
4
5 6
7 8 9 10 11 12 13
Marangos PJ, Schmechel D, Zis AP, Goodwin FK. The existence and neurobiological significance of neuronal and glial forms of the glycolytic enzyme enolase. Biol Psychiatr 1979;14:563-579. Sheppard MN, Corrin B, Bennet MH, Marangos PJ, Bloom SR, Polak JM. Immunocytochemical localization of neurone specific enolase in small cell carcinomas and carcinoid tumours of the lung. Histopathology 1984;8:171-181. Schurmann G, Betzler M, Buhr HJ. Chromogranin A, neuron-specific enolase and synaptophysin as neuroendocrine cell markers in the diagnosis of tumours of the gastro-entero-pancreatic system. Eur J Surg Oncol 1990;16:298-303. Kloppel G, Girard J, Polak JM, Vaitukaitis JL, Kasper M, Heitz PHU. Alpha-human chorionic gonadotropin and neuron-specific enolase as markers for malignancy and neuroendocrine nature of pancreatic endocrine tumours. Cancer Detect Prevent 1983;6:161- 166. Tapia FJ, Barbosa AJA, Marangos PJ, Polak JM, Bloom SR, Dermody C. Neuron-specific enolase is produced by neuroendocrine tumours. Lancet 1981;1:808-8 I 1. Lloyd RV, Mervak T, Schmidt K, Wraner TFCS, Wilson BS. Immunohistochemical detection of chromogranin and neuron specific enolase in pancreatic endocrine neoplasms. Am J Surg Pathol 1984;8:607-614. Ishiguro Y, Kato K, Ito T, Horisaura M, Nagaya M. Enolase isozymes as markers for differential diagnosis of neuroblastoma, rhabdomyosarcoma and Wilm’s tumour. Gann 1984;75:53-60. Leff EL, Brooks JSJ, Trojanowski JQ. Expression of neurotilament and neuron-specific enolase in small cell tumors of skin using immunohistochemistry. Cancer 1985;56:625-631. Royds JA, Parsons MA, Rennie IG, Timperley WR, Taylor CB. Enolase isoenzymes in benign and malignant melanocytic lesions. Diag Histopathol 1982;5:175- 181. Nakajima T, Kameya T, Tsumuraya M, Shimosato Y, Kato K. Enolase distribution in human brain tumours, retinoblastomas and pituitary adenomas. Brain Res 1984;308:215-222. Camey and Teeling. Neuron-specific enolase: how useful as a cancer marker? Eur J Cancer Oncol 1986;24:825-828. Schmechel DE. y-Subunit of the glycolytic enzyme enolase: nonspecific or neuron specific? Lab Invest 1985;52:239-242. Cunningham RT, Johnston CF, Irvine GB, McIlrath EM, McNeil1 A, Buchanan KD. Development
131
18 19 20 21
22 23
24
25 26 27 28 29
30
of a radioimmonoassay for neurone specific enolase (NSE) and its application in the study of patients receiving intra hepatic arterial streptozotocin and floxuridine. Clin Chim Acta 1990;189:275-286. Carney DN, Ihde DC, Cohen MH et al. Serum Neuron-specific enolase: a marker for disease extent and response to therapy of small-cell lung cancer. Lancet 1982;1:583-585. Ariyoshi Y, Kato T, Ueda R et al. Biological and clinical implication of neuron-specific enolase and creatine kinase BB in small cell lung cancer. Jpn J Clin Oncol 1986;16(3):213-221. Akoun GM, Scarna HM, Milleron BJ, Benichou MP, Herman DP. Serum neuron-specific enolase. A marker for disease extent and response to therapy for small cell lung cancer. Chest 1985;1:39-43. Cooper EH, Splinter TAW, Brown DA, Meurs MF, Peake MD, Pearson SL. Evaluation of a radioimmunoassay for neuron spectic enolase in small cell lung cancer. Br J Cancer 1985;52:333-338. Esscher T, Steiholtz L, Bergh J, Nou E, Nilsson K, Pahlman S. Neuron specific enolase: a useful diagnostic serum marker for small cell carcinoma of the lung. Thorax 1985;40:85-90. Johnston DH, Marangos PJ, Forbes JT et al. Potential utility of serum neuron-specific enolase levels in small cell carcinoma of the lung. Cancer Res 1984;44:5409-5414. Fischbach W, Jany B. Neuron-specific enolase in the diagnosis and therapy monitoring of lung cancer: A camparison with CEA, TPA, ferritin and calcitonin. Int J Biol Markers 1986;1:129-136. Adewole IF and Newlands ES. Neuron-specific enolase (NSE) as a tumour marker and comparative evaluation with carcinoembryonic antigen (CEA) in small-cell lung cancer. Med Oncol Tumour Pharmacol 1987;4:1I - 15. Zeltzer PM, Marangos PJ, Evans AE, Schneider SL. Serum neuron-specific enolase in children with neuroblastoma. Relationship to stage and disease course. Cancer 1986;57:1230-1234. Grouzmann E, Gicquel C, Plouin PF, Schlumberger M, Comoy E, Bohuon C. Neuropeptide Y and neuron-specific enolase levels in benign and malignant pheochromocytomas. Cancer 1990;66:1833-1835. Prinz RA, Bermes EW, Kimmel JR, Marangos PJ. Serum markers for pancreatic islet cell and intestinal carcinoid tumours: a comparison of neuron-specific enolase P-human chorionic gonadotropin and pancreatic polypeptide. Surgery 1983;94: 1019- 1023. Prinz RA, Marangos PJ. Use of neuron-specific enolase as a serum marker for neuroendocrine neoplasms. Surgery 1982;92:887-889. Ishiguro Y, Kato K, Ito T, Nagaya M, Yamada N, Sugito T. Nervous system-specific enolase in serum as a marker for neuroblastoma. Pediatrics 1983;72;696-700. Thomas P, Battifora H, Manderino GL, Patrick J. A monoclonal antibody against neuron-specific enolase. Am J Clin Pathol 1987;88:146-152. Ariyoshi Y, Kato K, Ishiguro Y, Ota K, Sato T, Suchi T. Evaluation of serum neuron-specific enolase as a tumour marker for carcinoma of the lung. Gann 1983;74:219-225. Vinores SA, Bonnin JM, Rubenstein LJ, Marangos PJ. Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other tissues. Arch Pathol Lab Med 1984;108:536-540. Kornblatt MJ, Keller A, Legault-Demare L. Changes in expression of the (r(~form of enolase during neuroblastoma differentiation. J Neurochem 1983;41:1563-1568.
123
CCA 05438
Serum neurone-specific enolase levels in patients with neuroendocrine and carcinoid tumours Rodat T. Cunninghama, Colin F. Johnstona, G. Brent Irvineb and Keith D. Buchanana aDepartment of Medicine and bDivision of Biochemistry, The Queen> University of Belfast, Northern Ireland (UK)
(Received 16th March 1992; revision received 29 September 1992; accepted 12th October 1992)
Key wora!s: Neurone-specific enolase; Neuroendocrine
tumours; Carcinoid tumours
We have examined concentrations of neurone-specific enolase (NSE) in sera from 18 patients with various neuroendocrine tumours, 26 patients with carcinoid tumours, 21 patients with non-neuroendocrine tumours and 37 control individuals. No statistically significant difference between the concentrations in patients with neuroendocrine tumours and patients with carcinoid tumours was found. However the NSE concentrations in patients with carcinoid and neuroendocrine tumours, when these two groups were combined, were significantly different from the patients with non-neuroendocrine tumours or the control individuals (P I 0.01). 38.5% of the patients with carcinoid tumours had raised NSE concentrations in serum; 55.5% of those with non carcinoid neuroendocrine tumours had raised concentrations. There appeared to be no correlation between the NSE concentrations and the extent of metastases.
Introduction Neurone-specific enolase (NSE, EC 4.2.1 .l 1) is an isozyme of the glycolytic enzyme, enolase. The enzyme occurs predominantly in neurones but has also been located in neuroendocrine cells [ 11. Correspondence to: Dr. R.T. Cunningham, Department of Medicine, Wellcome Research Laboratories, Grosvenor Road, Belfast, BT12 6BJ, UK.
124
Neoplasms of neuroendocrine cells are often associated with an overproduction of a hormone which in turn gives rise to a clinical syndrome. Zollinger Ellison Syndrome, for example is associated with overproduction of gastrin, while carcinoid syndrome is associated with 5hydroxytryptamine and tachykinin release. Some neuroendocrine tumours, however, may be clinically ‘silent’, possibly because a peptide is not secreted, or is abnormal, or not associated with a syndrome. Alternatively, the target cell receptors for that hormone may be abnormal, or an inhibitor may be co-secreted. The proposal that NSE would act as a universal marker for all neuroendocrine tumours including ‘silent’ tumours has resulted in immunocytochemical (ICC) studies in small cell lung cancer [2], tumours of the gastrointestinal tract [3-61, neuroblastomas [7], melanomas [8,9], pituitary adenomas [lo], etc. However non specific results have led to some debate about the reliability of ICC staining for NSE [11,1.2]. It is unclear whether these results are a consequence of using non-specific polyclonal antibodies [ 111. In addition to ICC studies there have been several assays developed to measure NSE concentrations in serum and plasma [13-281. This paper documents the results obtained when a radioimmunoassay (RIA) was used to measure NSE concentrations in sera from patients with neuroendocrine tumours. Patients and Methods Blood samples were collected from 18 patients with histologically confirmed noncarcinoid neuroendocrine tumours and from 26 patients with carcinoid tumours (Tables I and II, respectively). A carcinoid tumour was identified as being a tumour of neuroendocrine origin which was also chromogranin A and serotonin positive. The ages of the groups were 52.1 i 14.1 and 57.5 f 10.2 y, respectively and the groups were 10 males, 8 females and 16 males and 10 females, respectively. Twentytwo of the patients with carcinoid tumours had metastases and 10 of the patients with other non-carcinoid neuroendocrine tumours also had metastases. Of those patients with carcinoid tumours, 4 had suffered from carcinoid crisis. Blood samples were also collected from 21 patients (Table III) with tumours which were not of the neuroendocrine system, also confirmed by a lack of staining for serotonin and chromogranin A. The ages of these patients were 62.7 f 12.3 y and the group was 3 males and 18 females. A group of 37 control individuals (19 female and 18 male) was studied. They had cardiac or respiratory disease and were free from neuroendocrine or neurological disorders. None of these patients had any known malignant disease. The mean age in this group was 50.3 f 16.0 y. Blood samples were obtained from fasted individuals, 10 ml being withdrawn from the antecubital vein. Samples were then centrifuged at 500 x g for 20 min at 4°C and the serum was divided into 0.5-ml portions before being stored at -20°C prior to assay. Measurement of serum NSE
The assay for NSE was as previously reported [ 131.NSE was purified from human brain obtained less than 12 h post mortem and specific antibodies raised to the pro-
125
TABLE I Serum NSE levels in patients with neuroendocrine tumours No Sex
Age
Nature of tumour
Tumour burden
Primary site
Sites of metastases
1 M 2F
66 66
Gastrinoma MEA type I
I+ I+
None None
23 16
3
M
39
Gastrinoma
2+
Pancreas Pancreas/ parathyroid Pancreas
I3
4
F
72
2+++
Pancreas
5 6 7 8 9
M M M F M
51 67 55 51 67
2+ I 2+++ 0 2+++
10
M
29
Gastrinomal insulinoma/ VlPoma VIPoma lnsulinoma Unknown MCT Gastrinoma MCT Unknown
Lymph nodes Liver
Pancreas Pancreas Pancreas Thyroid Thyroid/ pancreas Pancreas
II
F
53
2++
12 13
F M
46 65
VlPomal WDHA Gastrinoma Gastrinoma
14 15 I6
55 35 23 44
I8
M
54
Gastrinoma Gastrinoma Unknown Glucagonomti gdstrinoma Unknown
0
11
F F M F
I I+ 2+++
Pancreas/ liver Unknown Pancreas/ liver Pancreas Unknown Unknown Pancreas
2++
Pancreas
I 2+++
Liver None Liver None Liver Liver/ lymph nodes Liver
NSE Pgll
II
16 13 21 28 II3 24
28
None Liver
5 3
None None None Liver
1
5 6 63
Liver
25
MEA, multiple endocrine adenoma; VIP, vasoactive intestinal peptide; MCT, medullary carcinoma of the thyroid.
tein. The protein was also radioactively labelled and used in the development and validation of a RIA to NSE. Separation of bound and free protein was by the second antibody separation technique. The assay sensitivity was 1 pgll. Intra assay variation was 11.0% at 46.6 i 1.7 &l ; 6.7% at 28.8 f 1.9 &l and 7.4% at 19.7 f 1.4 pg/l. Interassayvariationwas 13.9%at81 f 11.3&l;8.2%at 10.2 f 0.8&land 13.5% at 9.5 f 1.2 &l. Each value obtained was the mean of at least 6 duplicates. Statistical analysis
Serum NSE concentrations were compared between the patients with carcinoids and other non-carcinoid neuroendocrine tumours using the Mann-Whitney U-test. The same test was also used to compare NSE concentrations in those patients with neuroendocrine tumours (including those with carcinoids), in control individuals and in patients with non-neuroendocrine tumours.
126 TABLE II Serum NSE levels in patients with carcinoid tumours No. Sex
Age
Tumour burden
Carcinoid crisis
Primary site
Metastases (sites)
NSE ati
Liver Liver/ omentum Liver Liver Liver
18 30
1 2
M M
56 64
2 +++ 2 +++
N N
Ileum Unknown
3 4 5
M M F
60 45 62
2 +++ 2 +++ 2 +++
N Y N
6 7 8 9 10 11 12 13 14 15 16 17 18
M F M F M M F M F F M F M
59 59 58 19 59 72 71 44 49 74 56 58 58
2 +++ 2 +++ 0 2+ 2 +++ 2+ 2 ++ 0 0 2+ 2 ++ 2 +++ 2 +++
N N N N
Y
Lung Lung Lung/ pancreas Ileum Ileum Ileum Ileum Ileum Ileum Unknown Caecum Appendix Appendix Lung Ileum Lungliver
19 20
M F
33 52
2 ++ 2 ++
Y N
Unknown Ileum
21
M
59
2 +++
N
22 23 24 25
M F M M
46 44 55 60
2 +++
N N
2 +++ 2 ++
Y
26
F
62
Ileum/ pituitary/ liver Lung Lung Unknown Ileum/ liver Ileum
N N N N N N N N
N N
Liver Liver Liver Liver Liver Liver Liver None None Liver Liver Liver Liver/ hilar node/ bone marrow Liver Liver/ lymph nodes Liver
8 343 13 13 16 32 7 10 10 9 7 21 7 10 19 10
34 14 8
Liver None Liver Liver
570 17 11 5
None
8
Rt%UItS
Control individuals
Concentrations of 9.1 f 2.5 &l (mean f S.D.) were recorded for our 37 control individuals. This results in an upper limit of the reference of 14.1 pgA(9.1 f 2 S.D.). In the patients with non-neuroendocrine tumours, the concentration was 9.4 f 2.9 g/l. Two patients had NSE concentrations just above the upper limit of the reference interval (16.2 and 15.6 &l).
127 TABLE III Serum NSE levels in patients with non-neuroendocrine
tumours
Sex
Age
Tumour type
Primary Site
Site of metastases
NSE Wll
M F M M F
64 77 46 75 47
Lung Colon Colon Colon Breast
Liver Liver Liver Liver None
8.7 13.0 8.5 16.2 7.2
F
56
Neck
None
9.2
F F F F F F F F
93 56 69 64 80 60 44 54
Vagina Breast Cervix Oral cavity Nose Endometrium Cervix Neck
None Bone None None None None None None
6.3 10.8 10.8 6.8 15.6 7.4 10.2 8.1
F F
60 66
Breast Unknown
Bone Bone
8.4 7.2
F
63
Breast
Bone
9.3
F F F F
71 68 50 54
Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Infiltrating duct cell Non-Hodgkins lymphoma Squamous cell Squamous cell Squamous cell Squamous cell Squamous cell Adenocarcinoma Squamous cell Non-Hodgkins lymphoma Adenocarcinoma Clear cell carcinoma Invasive lobular cell Squamous cell Squamous cell Squamous cell Adenocarcinoma
Cervix Cervix Cervix Breast
None None None Bone
6.4 10.5 5.4 11.6
Neuroendocrine tumour-bearing patients
Serum NSE levels were above the upper limit of normal in 38.5% of subjects studied with carcinoid tumours. Serum NSE values within these carcinoid patients varied from 5 to 570 pg/l. Twenty-two of the patients with carcinoid tumours had liver metastases but only 8 of these had a serum NSE above normal. Only 4 of the carcinoid patients had suffered from carcinoid crisis and two of these had a normal serum NSE value at the time of assay. Of the remaining 22 carcinoid patients, 14 had a normal NSE serum concentration. In patients with non-carcinoid neuroendocrine tumours, NSE was elevated in 55.5% of the cases studied. Ten of these patients had secondary tumours on either the liver or lymph nodes. Of the 10 patients with metastases, 7 had elevated serum NSE concentrations. Of the 8 patients with no metastases, 5 had normal serum NSE values.
128
Statistical analysis, using the Mann-Whitney U-test, revealed no significance when NSE levels in patients with carcinoid tumours were compared with those levels in patients with non-carcinoid neuroendocrine tumours. However, when both of these two groups were combined and compared with levels obtained in patients with nonneuroendocrine tumours, a significant difference was recorded (P = 0.01). Levels in both groups combined were also significantly different from those obtained from normal individuals (P = 0.001). In 4 patients who had suffered carcinoid crisis, only two had elevated serum NSE. Metastases in carcinoid patients did not correlate with serum NSE value; 2 of 4 patients with no metastases were associated with high serum NSE concentrations, while other patients with metastases had levels within the normal range. Similarly serum NSE concentration in each of these patients showed no correlation with degree of tumour burden. Neither did tumour burden correlate with serum NSE concentration in patients with non-carcinoid neuroendocrine tumours (Table I) and some of these patients who had metastases had normal serum NSE concentrations. Discussion It was observed that 38.5% of patients with carcinoid tumours and 55.5% of patients with other neuroendocrine tumours had elevated serum NSE concentrations. In total 45.5% of all patients with a neuroendocrine tumour at diagnosis had an elevated serum NSE concentration. Previous studies have looked at serum NSE in a range of neuroendocrine tumours. In patients with small cell cancer of the lung at diagnosis, elevated serum NSE has been recorded in 69%, 71.4%, 65.1%, 68%, 72%, 73%, 60% and 76.5% of all patients studied [14-211. Elevated serum NSE concentrations have also been noted in patients with neuroblastoma [7,22] and in patients with phaeochromocytoma [23]. Prinz et al. [24] studied patients with pancreatic islet cell cancers and intestinal carcinoid tumours and found elevated serum NSE in 46% of all patients studied. This compares favourably with our value of 45.5% of all patients studied with neuroendocrine tumours. Marangos and Prinz also reported that elevated serum NSE concentrations were less likely in patients with intestinal carcinoids than in patients with other neuroendocrine tumours, even if liver metastases were present. They found NSE to be elevated in 1 of 5 patients with intestinal carcinoid, while we found that 4 of 12 patients known to have ideal carcinoids had elevated serum NSE. Prinz and Marangos [25] found that 9 of 21, or 42.9% of the APUDoma patients they studied, had clearly elevated NSE concentrations and that clear elevations were only observed in patients with extensive disease. Close correlation of serum NSE concentrations and disease extent has been noted by others [14,16,17,23] and it has been suggested as a useful prognostic indicator of disease progress in patients with a range of neuroendocrine tumours [19,20]. In neuroblastomas it has been reported that 79% of patients with a serum NSE concentration less than 100 ng/rnl survived 2 years after diagnosis [22]. In addition it was noted that serum NSE concentration reflected the stage of disease with patients in stage I having considerably lower concentrations than those in stage IV. It was noted that serum NSE concentrations in patients with stage IVs disease, which is prone to spontaneous regression and therefore clinically more benign had lower
129
concentrations than those in stage IV despite heavy tumour burden. Our results did not indicate a correlation between serum NSE concentration and disease extent since some of the patients studied had low concentrations of serum NSE, despite heavy tumour burden. However Prinz and Marangos [25] also recorded normal concentrations of serum NSE in patients with neuroendocrine tumours and metastases. This may be because NSE is only released as a result of cell lysis and that in these patients, the tumour cells are healthy. All the blood samples collected from the patients studied here were collected prior to cytotoxic chemotherapy, although some patients (4, 5, 7, 11, 17 from Table I and 1-6, 15-21, 24 and 25 from Table II) were taking the long-acting somatostatin analogue, octapeptide SMS 201-995 (Sandoz AG, Basel, Switzerland). This drug relieves the symptoms of carcinoid syndrome and functioning neuroendocrine tumours, presumably by inhibiting peptide synthesis or release. Of these 20 patients on SMS 201-995, only 10 had serum NSE concentrations above the upper limit of normal. We have found previously that the circulating concentrations of NSE are dramatically elevated after intra hepatic arterial streptozotocin and floxuridine [ 131. It may be possible for healthy tumour cells to be associated with normal serum NSE concentrations, although necrotic areas are more likely in larger tumours. Two carcinoid patients studied here who had suffered carcinoid crisis had greatly elevated concentrations of serum NSE, although two similar patients had normal serum NSE concentrations. Patients 4, 19 and 25 had blood samples collected 15, 3 and 28 days before carcinoid crisis and serum NSE concentrations of 343, 10 and 11 pg/l, respectively. Blood was collected 6 days after crisis in patient 20 who had a serum NSE of 34 &l. It may be that crisis is associated with the onset of the release of the contents of the neuroendocrine tumour resulting in subsequent flushing, lacrimation, diarrhoea, etc. If this release of contents involved cell breakdown then NSE should also be released. However if NSE has a serum half-life of 48 h as reported by Ishiguro et al. [26], this would imply the need to obtain a blood sample from the patient within this period. This might account for the normal serum NSE values in two of the patients who had previously had carcinoid crisis. Alternatively, the tumour cells may release their contents but remain viable. There have been reports concerning the non-specificity of NSE with elevated serum concentrations reported in patients with tumours which are not of the neuroendocrine system [ 11,121. We found that concentrations were very slightly elevated in 2 of 21 patients with non-neuroendocrine tumours but were still less than the mean normal value f 3 S.D. Ishiguro [7] has suggested that the elevated serum NSE concentrations he has reported in 2 of 6 patients with Wilm’s tumour may be as a result of the tendency for the undifferentiated small cells present to develop into a particular cell type, e.g. smooth or striated muscle. Carney and Teeling [ 1l] have suggested that serum NSE is neither sensitive or specific for small cell lung cancer although they do suggest that it may be of use in monitoring disease progress after chemotherapy and in distinguishing paediatric tumours. They also question whether the non-specific staining obtained in immunocytochemical studies of neuroendocrine tumours may be as a result of technical problems or problems with the antibody. Thomas et al. [27] have reported a much more specific monoclonal antibody to NSE with only 2% of non-neuronal, non-neuroendocrine tumours staining positively with respect to 20% non-specific staining using a polyclonal antibody.
130
Other factors which may account for nonspecific staining in non-neuronal, nonneuroendocrine tumours includes incorrect classification of the tumour type [28], altered metabolism [29] or aberrant gene expression [ 121. There have also been reports of transformation of one cell type to another [12,30]. Whatever the reasons for non-specific staining with NSE, it remains that immunhistochemically positive results should be treated with caution. This does not however preclude studies into the viability of NSE as a serum marker especially in monitoring chemotherapy [13,17,19,25]. In conclusion, our results show that serum NSE concentrations may be elevated in patients with neuroendocrine tumours, including carcinoid tumours but that they are not always raised. Although serum NSE may not be a particularly sensitive marker for the diagnosis of neuroendocrine tumours, despite reports to the contrary, it appears to be specific. Acknowledgements This work was supported in part by the Medical Research Council, UK and by the Northern Ireland Chest, Heart and Stroke Association. References 1 2
3
4
5 6
7 8 9 10 11 12 13
Marangos PJ, Schmechel D, Zis AP, Goodwin FK. The existence and neurobiological significance of neuronal and glial forms of the glycolytic enzyme enolase. Biol Psychiatr 1979;14:563-579. Sheppard MN, Corrin B, Bennet MH, Marangos PJ, Bloom SR, Polak JM. Immunocytochemical localization of neurone specific enolase in small cell carcinomas and carcinoid tumours of the lung. Histopathology 1984;8:171-181. Schurmann G, Betzler M, Buhr HJ. Chromogranin A, neuron-specific enolase and synaptophysin as neuroendocrine cell markers in the diagnosis of tumours of the gastro-entero-pancreatic system. Eur J Surg Oncol 1990;16:298-303. Kloppel G, Girard J, Polak JM, Vaitukaitis JL, Kasper M, Heitz PHU. Alpha-human chorionic gonadotropin and neuron-specific enolase as markers for malignancy and neuroendocrine nature of pancreatic endocrine tumours. Cancer Detect Prevent 1983;6:161- 166. Tapia FJ, Barbosa AJA, Marangos PJ, Polak JM, Bloom SR, Dermody C. Neuron-specific enolase is produced by neuroendocrine tumours. Lancet 1981;1:808-8 I 1. Lloyd RV, Mervak T, Schmidt K, Wraner TFCS, Wilson BS. Immunohistochemical detection of chromogranin and neuron specific enolase in pancreatic endocrine neoplasms. Am J Surg Pathol 1984;8:607-614. Ishiguro Y, Kato K, Ito T, Horisaura M, Nagaya M. Enolase isozymes as markers for differential diagnosis of neuroblastoma, rhabdomyosarcoma and Wilm’s tumour. Gann 1984;75:53-60. Leff EL, Brooks JSJ, Trojanowski JQ. Expression of neurotilament and neuron-specific enolase in small cell tumors of skin using immunohistochemistry. Cancer 1985;56:625-631. Royds JA, Parsons MA, Rennie IG, Timperley WR, Taylor CB. Enolase isoenzymes in benign and malignant melanocytic lesions. Diag Histopathol 1982;5:175- 181. Nakajima T, Kameya T, Tsumuraya M, Shimosato Y, Kato K. Enolase distribution in human brain tumours, retinoblastomas and pituitary adenomas. Brain Res 1984;308:215-222. Camey and Teeling. Neuron-specific enolase: how useful as a cancer marker? Eur J Cancer Oncol 1986;24:825-828. Schmechel DE. y-Subunit of the glycolytic enzyme enolase: nonspecific or neuron specific? Lab Invest 1985;52:239-242. Cunningham RT, Johnston CF, Irvine GB, McIlrath EM, McNeil1 A, Buchanan KD. Development
131
18 19 20 21
22 23
24
25 26 27 28 29
30
of a radioimmonoassay for neurone specific enolase (NSE) and its application in the study of patients receiving intra hepatic arterial streptozotocin and floxuridine. Clin Chim Acta 1990;189:275-286. Carney DN, Ihde DC, Cohen MH et al. Serum Neuron-specific enolase: a marker for disease extent and response to therapy of small-cell lung cancer. Lancet 1982;1:583-585. Ariyoshi Y, Kato T, Ueda R et al. Biological and clinical implication of neuron-specific enolase and creatine kinase BB in small cell lung cancer. Jpn J Clin Oncol 1986;16(3):213-221. Akoun GM, Scarna HM, Milleron BJ, Benichou MP, Herman DP. Serum neuron-specific enolase. A marker for disease extent and response to therapy for small cell lung cancer. Chest 1985;1:39-43. Cooper EH, Splinter TAW, Brown DA, Meurs MF, Peake MD, Pearson SL. Evaluation of a radioimmunoassay for neuron spectic enolase in small cell lung cancer. Br J Cancer 1985;52:333-338. Esscher T, Steiholtz L, Bergh J, Nou E, Nilsson K, Pahlman S. Neuron specific enolase: a useful diagnostic serum marker for small cell carcinoma of the lung. Thorax 1985;40:85-90. Johnston DH, Marangos PJ, Forbes JT et al. Potential utility of serum neuron-specific enolase levels in small cell carcinoma of the lung. Cancer Res 1984;44:5409-5414. Fischbach W, Jany B. Neuron-specific enolase in the diagnosis and therapy monitoring of lung cancer: A camparison with CEA, TPA, ferritin and calcitonin. Int J Biol Markers 1986;1:129-136. Adewole IF and Newlands ES. Neuron-specific enolase (NSE) as a tumour marker and comparative evaluation with carcinoembryonic antigen (CEA) in small-cell lung cancer. Med Oncol Tumour Pharmacol 1987;4:1I - 15. Zeltzer PM, Marangos PJ, Evans AE, Schneider SL. Serum neuron-specific enolase in children with neuroblastoma. Relationship to stage and disease course. Cancer 1986;57:1230-1234. Grouzmann E, Gicquel C, Plouin PF, Schlumberger M, Comoy E, Bohuon C. Neuropeptide Y and neuron-specific enolase levels in benign and malignant pheochromocytomas. Cancer 1990;66:1833-1835. Prinz RA, Bermes EW, Kimmel JR, Marangos PJ. Serum markers for pancreatic islet cell and intestinal carcinoid tumours: a comparison of neuron-specific enolase P-human chorionic gonadotropin and pancreatic polypeptide. Surgery 1983;94: 1019- 1023. Prinz RA, Marangos PJ. Use of neuron-specific enolase as a serum marker for neuroendocrine neoplasms. Surgery 1982;92:887-889. Ishiguro Y, Kato K, Ito T, Nagaya M, Yamada N, Sugito T. Nervous system-specific enolase in serum as a marker for neuroblastoma. Pediatrics 1983;72;696-700. Thomas P, Battifora H, Manderino GL, Patrick J. A monoclonal antibody against neuron-specific enolase. Am J Clin Pathol 1987;88:146-152. Ariyoshi Y, Kato K, Ishiguro Y, Ota K, Sato T, Suchi T. Evaluation of serum neuron-specific enolase as a tumour marker for carcinoma of the lung. Gann 1983;74:219-225. Vinores SA, Bonnin JM, Rubenstein LJ, Marangos PJ. Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other tissues. Arch Pathol Lab Med 1984;108:536-540. Kornblatt MJ, Keller A, Legault-Demare L. Changes in expression of the (r(~form of enolase during neuroblastoma differentiation. J Neurochem 1983;41:1563-1568.
