Neurol Sci DOI 10.1007/s10072-015-2190-5
ORIGINAL ARTICLE
The diagnostic value of minor salivary gland biopsy in clinically diagnosed patients with Parkinson’s disease: comparison with DAT PET scans Liyan Gao1,2,3 • Huimin Chen1,2,3 • Xin Li1,2,3 • Fangfei Li1,2,3 • Qiaohong Ou-Yang4 Tao Feng1,2,3
•
Received: 31 January 2015 / Accepted: 20 March 2015 Ó Springer-Verlag Italia 2015
Abstract To investigate the predictive value of minor salivary gland biopsy in clinically diagnosed early stage Parkinson’s disease (PD) patients, and to provide more evidence of minor salivary gland biopsy as a pathological diagnostic biomarker of PD. Thirteen patients with early stage PD and 13 age-matched controls were recruited. Hoehn and Yahr stage and Unified Parkinson’s disease Rating Scale Part III were employed to evaluate their severity of the disease. All the subjects underwent minor salivary gland biopsy and 11C-methyl-N-2b-carbomethoxy3b-(4-fluorophenyl) tropane (11C-CFT) DAT-PET scan. Immunohistochemical staining for Lewy-type alpha-synucleinopathy using antibody against alpha-synuclein (a-Syn) was performed in the tissues obtained from minor salivary gland. Abnormal accumulation of a-Syn was found around the gland cells in 9 of the 13 patients with PD, but in none of the control subjects. The a-Syn immunoreactive structures were located in the periacinar space. Twelve clinically diagnosed PD patients showed asymmetrical and relatively severe reduction of 11C-CFT uptake in the posterior putamen compared with the control. The sensitivity, & Tao Feng [email protected] 1
Center for Neurodegenerative Diseases, Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing 100050, China
2
China National Clinical Research Center for Neurological Diseases, Beijing, China
3
Parkinson’s Disease Center, Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China
4
Department of Nuclear Medicine, No. 1 Hospital, Affiliated to General Hospital of the Chinese People’s Liberation Army, Beijing, China
specificity, positive predictive value and negative predictive value of minor salivary gland biopsy were 75, 100, 100 and 25%, respectively, when compared with the DAT-PET imaging. Our results suggest that minor salivary gland biopsy does not hold high diagnostic accuracy as DATPET, but still has the potential to be a useful pathologic biomarker for PD, which is worth more investigations. Keywords Parkinson’s disease Alpha-synuclein Minor salivary gland biopsy Positron emission tomography
Introduction As a common neurodegenerative disease, Parkinson’s disease (PD) is clinically diagnosed relying mainly on the cardinal motor signs [1, 2]. Despite the fact that brain biopsy provides relatively accurate diagnosis and is regarded as the gold standard, yet it’s invasive and not accepted by most patients. From a pathological point of view, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies and Lewy neurites [3], in which the key component is aggregated alpha-synuclein (a-Syn) [4]. Recently, searching for a biomarker including Lewy pathology in regions of the peripheral organs or body fluids, such as skin, enteric nervous system, plasma and others [5–15], has raised much more concern. The presence of Lewy pathology in the minor salivary glands of patients with PD was considered as a possible biomarker of PD, but the results remains controversial in two studies. For instance, a-Syn has been detected in about 66 % of PD cases in a pilot study while merely in about 19 % of PD patients in another study [14, 15]. As a result, inadequate evidence was provided to assess this new diagnostic tool.
123
Neurol Sci
DAT imaging, is sensitive to detect presynaptic dopamine neuronal dysfunction so as to be considered as one of the useful diagnostic tool for degenerative Parkinsonism. To some extent, it was chosen as a neuroimaging biomarker. As reported, PET was described as 84 % sensitivity, 97 % specificity, 98 % positive predictive value (PPV), and 82 % negative predictive value (NPV) in the diagnosis of PD [16]. Despite the fact that DAT imaging improved the diagnosis of PD comparing to clinical diagnosis, its popularization was impeded due to high cost. Therefore, other biamarkers waits to be discovered and comfirmed. Taken that, our study combined minor salivary gland biopsy with DAT-PET imaging study to compare their accuracy and to testify the consistency between a-synucleinopathy in minor salivary glands and dysfunction of nigrostriatal dopamine. The aim of our study was to investigate the predictive value of minor salivary gland biopsy for the DAT-PET scans in clinically diagnosed early stage PD patients, and to provide more evidence for minor salivary gland biopsy as a pathologically diagnostic biomarker for PD.
surgeon who was blinded to the diagnosis of the subjects. The samples were fixed in formalin for 24 h, and then embedded in paraffin. Serial sections, 5 lm thick, were cut and prepared for hematoxylin. All sections were deparaffinized firstly and then blocked with 3 % H2O2 for 10 min to inhibit endogenous peroxidase. Antigen retrieval was carried out by incubation in citrate buffer for 30 min at 96–100 °C. After incubating in 2 % normal goat serum for 30 min, the sections were incubated in an anti-a-Syn monoclonal antibody that recognized multiple binding sites at amino acids 115–121 (3D5, 1:500 dilution; synthesized in-house [18] ) overnight. After removing excess antibody, and the Simplified HRP Rabbit/ Mouse kit (D30-1/D31-1; 1:200 dilution; GBI Labs, Mukilteo, WA, USA) was used according to the manufacturer’s instructions. After a final wash in 0.05 mol/ L Tris buffer (pH 7.6), a-Syn was visualized with 3,30 diaminobenzidine tetrahydrochloride in Tris buffer with added H2O2. After immunostaining, all sections were observed and photographed under an optical microscope (BX3-CBH; Olympus Corporation, Tokyo, Japan). 11
Materials and methods Patients We recruited 13 patients with sporadic PD in the early clinical stages (H–Y stage 1–2) and 13 age-matched control subjects without neurological or psychiatric diseases. PD patients were diagnosed according to the criteria of the United Kingdom Parkinson’s Disease Society Brain Bank [1]. Patients were recruited consecutively from the inpatient and outpatient clinics of Beijing Tiantan Hospital, Capital Medical University. The control subjects were recruited as volunteers from physical examination center or other departments of Beijing Tiantan hospital, with normal neurological statue and movement function, absence of neurological or psychiatric disease, as well as MMSE [24. Written informed consent was obtained either from the participants or their closest relatives. This study was performed in accordance with the Declaration of Helsinki and all procedures were approved and supervised by the ethics committee of Beijing Tiantan Hospital.
Methods Immunohistochemistry All minor salivary gland specimens were obtained according to standard procedures [17] by the same oral
123
C-CFT DAT-PET scan
In this study, we used DAT scan with 11C-CFT DAT PET (Center of No. 1 Hospital affiliated to General Hospital of the Chinese People’s Liberation Army). And this method has been described detailedly in our previous study [19]. Medication, (amantadine, levodopa, selegiline, pramipexole,piribedil,etc.) which could interfere with the radiotracer, was stopped at least 5 half-life times before the PET was made. PET scans were performed in a quiet and dimly lit room. Images were collected after intravenous injection of 185–370 MBq of 11C-CFT 40 min and continued 25–35 min to get tomography through computer reconstruction. A CT scan used for localization of the lesion site was performed firstly, and then a PET scan was carried out immediately. All the images acquired were reconstructed to get the transverse, sagittal, and coronal CT, PET, and PET/CT fusion images. Since no DAT distributes in cerebellum (CB), the CB was used as the reference area for region of interest (ROI) analysis. According to ROI and the same CT image, three most clear images were selected to outline the ROI of bilateral caudate nucleus, bilateral putamen and cerebellum. Software was used to get the average data. The average ROI was used to get (ROI-CB)/ CB as 11C-CFT uptake index of these areas. The index was further compared to that of healthy control to confirm the normality of tracer uptake. PET with asymmetrical low 11 C-CFT uptake was considered as positive result. The scans were analyzed by a nuclear medicine specialist who was blinded for the clinical diagnosis and the biopsy results.
Neurol Sci
Statistical analysis Statistical analysis was performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA), and p \ 0.05 was considered significant. Numeric data was expressed as the mean ± SD, or median and interquartile range, based on its distribution. The diagnostic accuracy of minor salivary gland biopsy was determined by comparing their results to DAT PET scans which is used as the surrogate gold standard. Diagnostic accuracy is defined as the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).
Results Demographic and clinical features All subjects underwent minor salivary gland biopsy without complications. The female/male ratio of PD patients was 5/8; the mean age of the patients was 56.29 ± 16.53 years; the mean disease duration was 4.58 ± 3.56 years with age at onset of 48.12 ± 16.68. The median H–Y stage was 2.0 (interquartile range 1.5–2.0); and the median UPDRS-III score was 27.5 (interquartile range 14.5–32.5). Details of the demographic and clinical characteristics of all PD cases are listed in Table 1. Two groups were age- and gender- matched. a-Syn expression in salivary glands All the immunostained slides were reviewed for the presence and intensity of a-Syn by two pathologists who were
blinded to the diagnosis. Alpha-synuclein immunoreactive structures were detected in the minor salivary glands in nine of the thirteen PD subjects, but no sample from controls showed negative staining. Further more, we found that the a-Syn inclusions were located in the periacinar space (Fig. 1). Predictive value of minor salivary gland biopsy for the results of the DAT PET scans The 11C-CFT uptake in the caudate, anterior and posterior putamen was reduced at different levels in 12 the PD patients. Twelve clinically diagnosed PD patients showed asymmetrical and relatively severe reduction compared with the controls. Twelve control subjects showed normal DAT PET scan and 1 displayed symmetrical and slight reduction of 11C-CFT uptake in bilateral posterior of putamen. The pathological biopsy result was in accordance with DAT-PET scan in 9 patients (Fig. 1). When comparing the imaging result, the sensitivity, specificity, PPV and NPV of our biopsy result were 75, 100, 100 and 25% respectively (Table 2).
Discussion As a kind of neurodegenerative disease, brain pathological examination detecting synucleinopathy is currently considered as the most accurate way for the diagnosis of PD, which yet is invasive in vivo and difficult for most patients to accept. More and more evidence shed light on the neuropathological process in peripheral organs which might make it possible to establish a substitute biopsiable
Table 1 Demographic and clinical features of PD cases UPDRS-III total score
a-Syn inclusions
DAT-PET
1.5
27.5
?
?
1.5
31
?
?
1
22
?
?
54
1.5
16
?
?
29
1.5
17
?
?
10 7
44 54
2 2
11 13
? -
? ?
75
1
74
2
28
?
?
M
75
1
74
2
28
?
?
M
33
2
31
2
41
-
?
M
33
7
26
1
13
-
?
12
F
71
12
59
2
35
?
?
13
F
55
4
51
2
34
-
-
PD patient number
Sex
Age
Disease duration (years)
Age of PD onset
1
F
57
1.5
55.5
2
F
55
5
50
3
M
26
2
24
4
F
56
2
5
M
34
5
6 7
M M
54 61
8
M
9 10 11
H–Y stage
?, positive; -, negative
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Neurol Sci Fig. 1 The minor salivary biopsy results compared with the DAT-PET imagine results of PD patients. a, c a-Syn inclusions were located in the periacinar space in minor salivary (940). b, d The radioactive uptake of 11C-CFT in bilateral putamina was reduced compared with the control mean
(A)
(B)
(C)
(D)
Table 2 Predictive value of minor salivary gland biopsy for the DAT-PET scans
Minor salivary gland biopsy
Sensitivity (%)
Specificity (%)
Positive predictive value (%)
Negative predictive value (%)
75
100
100
25
marker as we discussed in the introduction. In our study, we chose the minor salivary gland biopsy to detect the presence of a-Syn inclusions, where abnormal a-Syn accumulation occurred in 9 of the 13 PD subjects and the positive incidence was 68.9 %. Our result demonstrated the presence of a-Syn inclusions in the PD patients of H–Y stage 1–2, meaning probable capability of early detection of PD by this method. By contrast, it was reported in other two studies that alpha-synucleinopathy occurred in the submandibular gland of all the PD patients, which were autopsy-based and inevitably most PD patients were of H–Y stage 3–4. Thus, we postulate that a-Syn inclusions in the minor salivary gland biopsy might be an early but less sensitive biomarker of PD. In our study, we compared the pathological result with the PET imaging. As a powerful neuroimaging biomarker
123
of PD, PET has been rewarded for its high sensitivity and specificity for detecting striatal dopamine deficiency in PD patients. Investigation of DAT has been widely used and studied [20–22]. Kovacs et al. [23] reported that DAT immunoreactivity in the striatum of PD patients inversely correlated with the total alpha-synuclein burden in the substantia. In animal experiment, synuclein knockout mice showed increased dopamine release from dopaminergic synapses [24]. These results gave a hint about the correlation between imaging and clinicopathology of PD. Therefore, DAT PET and biopsy are reasonable to apply simultaneously. We chose 11C-CFT as the marker, which bound with DAT specifically with high binding efficiency. Although its use remains disputed and it is less accurate than post-mortem neuropathological analysis, it is still one of the best possible alternatives at present. And for the first time, we compared the pathological result in peripheral nervous system with neuroimaging result. In our study, the 11 C-CFT uptake in the caudate, anterior and posterior putamen was reduced at different levels in 12 the PD patients, including all the subjects in a-Syn inclusions positive group. By comparing with DAT PET imaging, minor salivary biopsy showed lower accuracy. When evaluating the predictive value of minor salivary gland biopsy adjusted to the results of the DAT PET scans, the sensitivity,
Neurol Sci
specificity, PPV and NPV of our biopsy result were 75, 100, 100 and 25%, respectively. The PPV of minor salivary biopsy against DAT PET in our study was as high as 100 %, which means patients with a positive pathological result in minor salivary gland has a relatively high probability to be diagnosed as PD accurately. We postulated that positive minor salivary biopsy might be a predictor of PD. However, our results demonstrated the low NPV of minor salivary biopsy against DAT PET. In other words, negative result in minor salivary biopsy could not rule out the diagnosis of PD. Consequently, we also postulated that the value of minor salivary biopsy is somehow limited, for it possibly misses out PD patients. Comprehending these upside and downside, minor salivary biopsy might be able to provide some reference in the clinical. Besides, the clinical use of DAT PET as a routine diagnostic tool for PD is limited, owing to its high cost and absence in most primary hospitals. Minor salivary gland biopsy that we chose is less invasive and relatively well tolerated. In our study, all subjects underwent minor salivary gland biopsy without complications. This tool is safe and has a wider usage for diagnostic purposes in other diseases, including sjogren syndrome. In conclusion, minor salivary biopsy might be a cheap and helpful tool in clinical diagnosis of early stage PD, which also displayed unsatisfactory NPV. In present study, 12 control subjects showed normal DAT PET scan and 1 displayed symmetrical and slight reduction of 11C-CFT uptake in bilateral posterior of putamen. Such result of DAT PET scan has been found in normal elders previously as well. This subject was a 71-year-old male who was absent from any motor (bradykinesia, tremor, rigidity and gait disorders) or nonmotor symptoms (depression, olfactory disorders, rapid eye movement sleep behavior disorder, etc.). Conversely, 12 clinically diagnosed PD patients showed asymmetrical and relatively severe reduction compared with the control. The main difference of PET scan between PD and control was the type and level of the reduction of the uptake, although there is still no definite quantitative criterion of PET scan for distinguishing PD from the normal. In clinical work, experienced neurologists read the PET results and diagnose. So we regarded this subject as normal control. The present study has some limitations. We enrolled a small number of subjects, which should be expanded in the future. A further case–control study should be established to investigate a-Syn deposition in the minor salivary gland of PD and other diseases characterized by a-Syn pathology, such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Moreover, owing to the limited volume of gland tissue that we obtained, we were unable to carry out gel electrophoresis of a-Syn.
In conclusion, our results showed that a-Syn was present in the minor salivary gland of 69.2 % patients with early clinical stage PD, indicating it as a promising biomarker in peripheral nervous system of PD. Although minor salivary gland biopsy does not hold the diagnostic accuracy as high as DAT-PET, the PPV of minor salivary biopsy against DAT PET was high. It is also suggested that the combination of minor salivary gland and DAT PET scan would increase the accuracy of early diagnosis of idiopathic PD. Acknowledgments The authors wish to thank the patients who took part in the study. This study was sponsored by grants from the Beijing Municipal Science and Technology Commission, China (Grant No. Z111107058811012), the High Level Technical Personnel Training Project of Beijing Health System, China (Grant No. 2011-3-022), the Beijing Natural Science Foundation, Beijing Municipal Commission of Education, China (Grant No. kz20120025028), and the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant No. 2013BAI09B03). Conflict of interest
The authors declare no conflicts of interest.
References 1. Hughes AJ et al (1992) Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 55(3):181–184 2. Poewe W (2008) Non-motor symptoms in Parkinson’s disease. Eur J Neurol 15(Suppl 1):14–20 3. Yasuda T et al (2013) Neurodegenerative changes initiated by presynaptic dysfunction. Transl Neurodegener 2(1):16 4. Dickson DW et al (2009) Neuropathological assessment of Parkinson’s disease: refining the diagnostic criteria. Lancet Neurol 8(12):1150–1157 5. Lebouvier T et al (2010) Biopsable neural tissues: toward new biomarkers for Parkinson’s disease? Front Psychiatry 1:128 6. Michell AW, Luheshi LM, Barker RA (2005) Skin and platelet alpha-synuclein as peripheral biomarkers of Parkinson’s disease. Neurosci Lett 381(3):294–298 7. Miki Y et al (2010) Clinical availability of skin biopsy in the diagnosis of Parkinson’s disease. Neurosci Lett 469(3):357–359 8. Lebouvier T et al (2008) Pathological lesions in colonic biopsies during Parkinson’s disease. Gut 57(12):1741–1743 9. Lebouvier T et al (2010) Colonic biopsies to assess the neuropathology of Parkinson’s disease and its relationship with symptoms. PLoS One 5(9):e12728 10. Pouclet H et al (2012) A comparison between colonic submucosa and mucosa to detect Lewy pathology in Parkinson’s disease. Neurogastroenterol Motil 24(4):e202–e205 11. Shannon KM et al (2012) Alpha-synuclein in colonic submucosa in early untreated Parkinson’s disease. Mov Disord 27(6): 709–715 12. Del Tredici K et al (2010) Lewy pathology in the submandibular gland of individuals with incidental Lewy body disease and sporadic Parkinson’s disease. Acta Neuropathol 119(6):703–713 13. Beach TG et al (2013) Submandibular gland biopsy for the diagnosis of Parkinson disease. J Neuropathol Exp Neurol 72(2): 130–136 14. Cersosimo MG et al (2011) Alpha-synuclein immunoreactivity in minor salivary gland biopsies of Parkinson’s disease patients. Mov Disord 26(1):188–190
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Neurol Sci 15. Folgoas E et al (2013) Diagnostic value of minor salivary glands biopsy for the detection of Lewy pathology. Neurosci Lett 551:62–64 16. Tang CC et al (2010) Differential diagnosis of parkinsonism: a metabolic imaging study using pattern analysis. Lancet Neurol 9(2):149–158 17. Caporali R et al (2008) Safety and usefulness of minor salivary gland biopsy: retrospective analysis of 502 procedures performed at a single center. Arthritis Rheum 59(5):714–720 18. Yu S et al (2007) Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody. Neuroscience 145(2):539–555 19. Liu P, Li X, Li FF, Ou-Yang QH, Zhang HX et al (2014) The predictive value of transcranial sonography in clinically diagnosed patients with early stage Parkinson’s disease: comparison with DAT PET scans. Neurosci Lett 582:99–103
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20. Kassiou M et al (2009) Challenges in molecular imaging of Parkinson’s disease: a brief overview. Brain Res Bull 78(2–3): 105–108 21. Sioka C, Fotopoulos A, Kyritsis AP (2010) Recent advances in PET imaging for evaluation of Parkinson’s disease. Eur J Nucl Med Mol Imaging 37(8):1594–1603 22. Biju G, de la Fuente-Fernandez R (2009) Dopaminergic function and progression of Parkinson’s disease: PET findings. Parkinsonism Relat Disord 15(Suppl 4):S38–S40 23. Kovacs GG et al (2008) Nigral burden of alpha-synuclein correlates with striatal dopamine deficit. Mov Disord 23(11): 1608–1612 24. Senior SL et al (2008) Increased striatal dopamine release and hyperdopaminergic-like behaviour in mice lacking both alphasynuclein and gamma-synuclein. Eur J Neurosci 27(4):947–957
ORIGINAL ARTICLE
The diagnostic value of minor salivary gland biopsy in clinically diagnosed patients with Parkinson’s disease: comparison with DAT PET scans Liyan Gao1,2,3 • Huimin Chen1,2,3 • Xin Li1,2,3 • Fangfei Li1,2,3 • Qiaohong Ou-Yang4 Tao Feng1,2,3
•
Received: 31 January 2015 / Accepted: 20 March 2015 Ó Springer-Verlag Italia 2015
Abstract To investigate the predictive value of minor salivary gland biopsy in clinically diagnosed early stage Parkinson’s disease (PD) patients, and to provide more evidence of minor salivary gland biopsy as a pathological diagnostic biomarker of PD. Thirteen patients with early stage PD and 13 age-matched controls were recruited. Hoehn and Yahr stage and Unified Parkinson’s disease Rating Scale Part III were employed to evaluate their severity of the disease. All the subjects underwent minor salivary gland biopsy and 11C-methyl-N-2b-carbomethoxy3b-(4-fluorophenyl) tropane (11C-CFT) DAT-PET scan. Immunohistochemical staining for Lewy-type alpha-synucleinopathy using antibody against alpha-synuclein (a-Syn) was performed in the tissues obtained from minor salivary gland. Abnormal accumulation of a-Syn was found around the gland cells in 9 of the 13 patients with PD, but in none of the control subjects. The a-Syn immunoreactive structures were located in the periacinar space. Twelve clinically diagnosed PD patients showed asymmetrical and relatively severe reduction of 11C-CFT uptake in the posterior putamen compared with the control. The sensitivity, & Tao Feng [email protected] 1
Center for Neurodegenerative Diseases, Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing 100050, China
2
China National Clinical Research Center for Neurological Diseases, Beijing, China
3
Parkinson’s Disease Center, Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China
4
Department of Nuclear Medicine, No. 1 Hospital, Affiliated to General Hospital of the Chinese People’s Liberation Army, Beijing, China
specificity, positive predictive value and negative predictive value of minor salivary gland biopsy were 75, 100, 100 and 25%, respectively, when compared with the DAT-PET imaging. Our results suggest that minor salivary gland biopsy does not hold high diagnostic accuracy as DATPET, but still has the potential to be a useful pathologic biomarker for PD, which is worth more investigations. Keywords Parkinson’s disease Alpha-synuclein Minor salivary gland biopsy Positron emission tomography
Introduction As a common neurodegenerative disease, Parkinson’s disease (PD) is clinically diagnosed relying mainly on the cardinal motor signs [1, 2]. Despite the fact that brain biopsy provides relatively accurate diagnosis and is regarded as the gold standard, yet it’s invasive and not accepted by most patients. From a pathological point of view, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies and Lewy neurites [3], in which the key component is aggregated alpha-synuclein (a-Syn) [4]. Recently, searching for a biomarker including Lewy pathology in regions of the peripheral organs or body fluids, such as skin, enteric nervous system, plasma and others [5–15], has raised much more concern. The presence of Lewy pathology in the minor salivary glands of patients with PD was considered as a possible biomarker of PD, but the results remains controversial in two studies. For instance, a-Syn has been detected in about 66 % of PD cases in a pilot study while merely in about 19 % of PD patients in another study [14, 15]. As a result, inadequate evidence was provided to assess this new diagnostic tool.
123
Neurol Sci
DAT imaging, is sensitive to detect presynaptic dopamine neuronal dysfunction so as to be considered as one of the useful diagnostic tool for degenerative Parkinsonism. To some extent, it was chosen as a neuroimaging biomarker. As reported, PET was described as 84 % sensitivity, 97 % specificity, 98 % positive predictive value (PPV), and 82 % negative predictive value (NPV) in the diagnosis of PD [16]. Despite the fact that DAT imaging improved the diagnosis of PD comparing to clinical diagnosis, its popularization was impeded due to high cost. Therefore, other biamarkers waits to be discovered and comfirmed. Taken that, our study combined minor salivary gland biopsy with DAT-PET imaging study to compare their accuracy and to testify the consistency between a-synucleinopathy in minor salivary glands and dysfunction of nigrostriatal dopamine. The aim of our study was to investigate the predictive value of minor salivary gland biopsy for the DAT-PET scans in clinically diagnosed early stage PD patients, and to provide more evidence for minor salivary gland biopsy as a pathologically diagnostic biomarker for PD.
surgeon who was blinded to the diagnosis of the subjects. The samples were fixed in formalin for 24 h, and then embedded in paraffin. Serial sections, 5 lm thick, were cut and prepared for hematoxylin. All sections were deparaffinized firstly and then blocked with 3 % H2O2 for 10 min to inhibit endogenous peroxidase. Antigen retrieval was carried out by incubation in citrate buffer for 30 min at 96–100 °C. After incubating in 2 % normal goat serum for 30 min, the sections were incubated in an anti-a-Syn monoclonal antibody that recognized multiple binding sites at amino acids 115–121 (3D5, 1:500 dilution; synthesized in-house [18] ) overnight. After removing excess antibody, and the Simplified HRP Rabbit/ Mouse kit (D30-1/D31-1; 1:200 dilution; GBI Labs, Mukilteo, WA, USA) was used according to the manufacturer’s instructions. After a final wash in 0.05 mol/ L Tris buffer (pH 7.6), a-Syn was visualized with 3,30 diaminobenzidine tetrahydrochloride in Tris buffer with added H2O2. After immunostaining, all sections were observed and photographed under an optical microscope (BX3-CBH; Olympus Corporation, Tokyo, Japan). 11
Materials and methods Patients We recruited 13 patients with sporadic PD in the early clinical stages (H–Y stage 1–2) and 13 age-matched control subjects without neurological or psychiatric diseases. PD patients were diagnosed according to the criteria of the United Kingdom Parkinson’s Disease Society Brain Bank [1]. Patients were recruited consecutively from the inpatient and outpatient clinics of Beijing Tiantan Hospital, Capital Medical University. The control subjects were recruited as volunteers from physical examination center or other departments of Beijing Tiantan hospital, with normal neurological statue and movement function, absence of neurological or psychiatric disease, as well as MMSE [24. Written informed consent was obtained either from the participants or their closest relatives. This study was performed in accordance with the Declaration of Helsinki and all procedures were approved and supervised by the ethics committee of Beijing Tiantan Hospital.
Methods Immunohistochemistry All minor salivary gland specimens were obtained according to standard procedures [17] by the same oral
123
C-CFT DAT-PET scan
In this study, we used DAT scan with 11C-CFT DAT PET (Center of No. 1 Hospital affiliated to General Hospital of the Chinese People’s Liberation Army). And this method has been described detailedly in our previous study [19]. Medication, (amantadine, levodopa, selegiline, pramipexole,piribedil,etc.) which could interfere with the radiotracer, was stopped at least 5 half-life times before the PET was made. PET scans were performed in a quiet and dimly lit room. Images were collected after intravenous injection of 185–370 MBq of 11C-CFT 40 min and continued 25–35 min to get tomography through computer reconstruction. A CT scan used for localization of the lesion site was performed firstly, and then a PET scan was carried out immediately. All the images acquired were reconstructed to get the transverse, sagittal, and coronal CT, PET, and PET/CT fusion images. Since no DAT distributes in cerebellum (CB), the CB was used as the reference area for region of interest (ROI) analysis. According to ROI and the same CT image, three most clear images were selected to outline the ROI of bilateral caudate nucleus, bilateral putamen and cerebellum. Software was used to get the average data. The average ROI was used to get (ROI-CB)/ CB as 11C-CFT uptake index of these areas. The index was further compared to that of healthy control to confirm the normality of tracer uptake. PET with asymmetrical low 11 C-CFT uptake was considered as positive result. The scans were analyzed by a nuclear medicine specialist who was blinded for the clinical diagnosis and the biopsy results.
Neurol Sci
Statistical analysis Statistical analysis was performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA), and p \ 0.05 was considered significant. Numeric data was expressed as the mean ± SD, or median and interquartile range, based on its distribution. The diagnostic accuracy of minor salivary gland biopsy was determined by comparing their results to DAT PET scans which is used as the surrogate gold standard. Diagnostic accuracy is defined as the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).
Results Demographic and clinical features All subjects underwent minor salivary gland biopsy without complications. The female/male ratio of PD patients was 5/8; the mean age of the patients was 56.29 ± 16.53 years; the mean disease duration was 4.58 ± 3.56 years with age at onset of 48.12 ± 16.68. The median H–Y stage was 2.0 (interquartile range 1.5–2.0); and the median UPDRS-III score was 27.5 (interquartile range 14.5–32.5). Details of the demographic and clinical characteristics of all PD cases are listed in Table 1. Two groups were age- and gender- matched. a-Syn expression in salivary glands All the immunostained slides were reviewed for the presence and intensity of a-Syn by two pathologists who were
blinded to the diagnosis. Alpha-synuclein immunoreactive structures were detected in the minor salivary glands in nine of the thirteen PD subjects, but no sample from controls showed negative staining. Further more, we found that the a-Syn inclusions were located in the periacinar space (Fig. 1). Predictive value of minor salivary gland biopsy for the results of the DAT PET scans The 11C-CFT uptake in the caudate, anterior and posterior putamen was reduced at different levels in 12 the PD patients. Twelve clinically diagnosed PD patients showed asymmetrical and relatively severe reduction compared with the controls. Twelve control subjects showed normal DAT PET scan and 1 displayed symmetrical and slight reduction of 11C-CFT uptake in bilateral posterior of putamen. The pathological biopsy result was in accordance with DAT-PET scan in 9 patients (Fig. 1). When comparing the imaging result, the sensitivity, specificity, PPV and NPV of our biopsy result were 75, 100, 100 and 25% respectively (Table 2).
Discussion As a kind of neurodegenerative disease, brain pathological examination detecting synucleinopathy is currently considered as the most accurate way for the diagnosis of PD, which yet is invasive in vivo and difficult for most patients to accept. More and more evidence shed light on the neuropathological process in peripheral organs which might make it possible to establish a substitute biopsiable
Table 1 Demographic and clinical features of PD cases UPDRS-III total score
a-Syn inclusions
DAT-PET
1.5
27.5
?
?
1.5
31
?
?
1
22
?
?
54
1.5
16
?
?
29
1.5
17
?
?
10 7
44 54
2 2
11 13
? -
? ?
75
1
74
2
28
?
?
M
75
1
74
2
28
?
?
M
33
2
31
2
41
-
?
M
33
7
26
1
13
-
?
12
F
71
12
59
2
35
?
?
13
F
55
4
51
2
34
-
-
PD patient number
Sex
Age
Disease duration (years)
Age of PD onset
1
F
57
1.5
55.5
2
F
55
5
50
3
M
26
2
24
4
F
56
2
5
M
34
5
6 7
M M
54 61
8
M
9 10 11
H–Y stage
?, positive; -, negative
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Neurol Sci Fig. 1 The minor salivary biopsy results compared with the DAT-PET imagine results of PD patients. a, c a-Syn inclusions were located in the periacinar space in minor salivary (940). b, d The radioactive uptake of 11C-CFT in bilateral putamina was reduced compared with the control mean
(A)
(B)
(C)
(D)
Table 2 Predictive value of minor salivary gland biopsy for the DAT-PET scans
Minor salivary gland biopsy
Sensitivity (%)
Specificity (%)
Positive predictive value (%)
Negative predictive value (%)
75
100
100
25
marker as we discussed in the introduction. In our study, we chose the minor salivary gland biopsy to detect the presence of a-Syn inclusions, where abnormal a-Syn accumulation occurred in 9 of the 13 PD subjects and the positive incidence was 68.9 %. Our result demonstrated the presence of a-Syn inclusions in the PD patients of H–Y stage 1–2, meaning probable capability of early detection of PD by this method. By contrast, it was reported in other two studies that alpha-synucleinopathy occurred in the submandibular gland of all the PD patients, which were autopsy-based and inevitably most PD patients were of H–Y stage 3–4. Thus, we postulate that a-Syn inclusions in the minor salivary gland biopsy might be an early but less sensitive biomarker of PD. In our study, we compared the pathological result with the PET imaging. As a powerful neuroimaging biomarker
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of PD, PET has been rewarded for its high sensitivity and specificity for detecting striatal dopamine deficiency in PD patients. Investigation of DAT has been widely used and studied [20–22]. Kovacs et al. [23] reported that DAT immunoreactivity in the striatum of PD patients inversely correlated with the total alpha-synuclein burden in the substantia. In animal experiment, synuclein knockout mice showed increased dopamine release from dopaminergic synapses [24]. These results gave a hint about the correlation between imaging and clinicopathology of PD. Therefore, DAT PET and biopsy are reasonable to apply simultaneously. We chose 11C-CFT as the marker, which bound with DAT specifically with high binding efficiency. Although its use remains disputed and it is less accurate than post-mortem neuropathological analysis, it is still one of the best possible alternatives at present. And for the first time, we compared the pathological result in peripheral nervous system with neuroimaging result. In our study, the 11 C-CFT uptake in the caudate, anterior and posterior putamen was reduced at different levels in 12 the PD patients, including all the subjects in a-Syn inclusions positive group. By comparing with DAT PET imaging, minor salivary biopsy showed lower accuracy. When evaluating the predictive value of minor salivary gland biopsy adjusted to the results of the DAT PET scans, the sensitivity,
Neurol Sci
specificity, PPV and NPV of our biopsy result were 75, 100, 100 and 25%, respectively. The PPV of minor salivary biopsy against DAT PET in our study was as high as 100 %, which means patients with a positive pathological result in minor salivary gland has a relatively high probability to be diagnosed as PD accurately. We postulated that positive minor salivary biopsy might be a predictor of PD. However, our results demonstrated the low NPV of minor salivary biopsy against DAT PET. In other words, negative result in minor salivary biopsy could not rule out the diagnosis of PD. Consequently, we also postulated that the value of minor salivary biopsy is somehow limited, for it possibly misses out PD patients. Comprehending these upside and downside, minor salivary biopsy might be able to provide some reference in the clinical. Besides, the clinical use of DAT PET as a routine diagnostic tool for PD is limited, owing to its high cost and absence in most primary hospitals. Minor salivary gland biopsy that we chose is less invasive and relatively well tolerated. In our study, all subjects underwent minor salivary gland biopsy without complications. This tool is safe and has a wider usage for diagnostic purposes in other diseases, including sjogren syndrome. In conclusion, minor salivary biopsy might be a cheap and helpful tool in clinical diagnosis of early stage PD, which also displayed unsatisfactory NPV. In present study, 12 control subjects showed normal DAT PET scan and 1 displayed symmetrical and slight reduction of 11C-CFT uptake in bilateral posterior of putamen. Such result of DAT PET scan has been found in normal elders previously as well. This subject was a 71-year-old male who was absent from any motor (bradykinesia, tremor, rigidity and gait disorders) or nonmotor symptoms (depression, olfactory disorders, rapid eye movement sleep behavior disorder, etc.). Conversely, 12 clinically diagnosed PD patients showed asymmetrical and relatively severe reduction compared with the control. The main difference of PET scan between PD and control was the type and level of the reduction of the uptake, although there is still no definite quantitative criterion of PET scan for distinguishing PD from the normal. In clinical work, experienced neurologists read the PET results and diagnose. So we regarded this subject as normal control. The present study has some limitations. We enrolled a small number of subjects, which should be expanded in the future. A further case–control study should be established to investigate a-Syn deposition in the minor salivary gland of PD and other diseases characterized by a-Syn pathology, such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Moreover, owing to the limited volume of gland tissue that we obtained, we were unable to carry out gel electrophoresis of a-Syn.
In conclusion, our results showed that a-Syn was present in the minor salivary gland of 69.2 % patients with early clinical stage PD, indicating it as a promising biomarker in peripheral nervous system of PD. Although minor salivary gland biopsy does not hold the diagnostic accuracy as high as DAT-PET, the PPV of minor salivary biopsy against DAT PET was high. It is also suggested that the combination of minor salivary gland and DAT PET scan would increase the accuracy of early diagnosis of idiopathic PD. Acknowledgments The authors wish to thank the patients who took part in the study. This study was sponsored by grants from the Beijing Municipal Science and Technology Commission, China (Grant No. Z111107058811012), the High Level Technical Personnel Training Project of Beijing Health System, China (Grant No. 2011-3-022), the Beijing Natural Science Foundation, Beijing Municipal Commission of Education, China (Grant No. kz20120025028), and the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant No. 2013BAI09B03). Conflict of interest
The authors declare no conflicts of interest.
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