N-CAM expression: The study of muscle disease in a tertiary center of Thailand

The neural cell adhesion molecule (N-CAM), also called CD56, is a cell-surface glycoprotein that mediates intercellular adhesive interactions in the nervous system. N-CAM is expressed in neuromuscular endplates, nerves, satellite cells, and embryonic muscle, but it is lost as development proceeds and is nearly absent from adult muscle. N-CAM re-expression was detected in a denervated, regenerating and degenerated muscle bres. Muscle disease or myopathy is diagnosed based on the presence of denervated, regenerating and degenerated bres on muscle biopsy combined with clinical information. Some regenerating bres looked like normal bre and were challenging to identify but showed positive NCAM staining. To explore the expression of N-CAM in muscle disease.


Introduction
Muscle disease or myopathy is a neuromuscular disorder in which the primary symptom is muscle weakness due to dysfunction of muscle bre. The condition can be acquired, familial, and congenital disorders of skeletal muscle. Common muscle diseases include muscular dystrophy, congenital myopathy, mitochondrial myopathy, in ammatory myopathy, and neurogenic myopathy [1]. Diagnosis of muscle disease requires all modalities; clinical examination, serologic markers, electrodiagnostic test, pathologic ndings from the muscle biopsy specimen, and molecular genetic study to make a nal diagnosis [2,3]. Muscle biopsy is a useful tool for the diagnostic evaluation of muscle disease. Still, the pathological ndings can vary from subtle myopathic changes to normal [2,3]. Immunohistochemistry (IHC) is an ancillary tool in muscle diseases diagnosis [2,3,4]. The neural cell adhesion molecule (N-CAM), also called CD56, is a cell-surface glycoprotein that was discovered in the embryonic brain and which mediates intercellular adhesive interactions in the nervous system. It is also expressed in neuromuscular endplates, nerves, satellite cells, and embryonic muscle bres to regulate nerve-muscle interaction [5,6,7,8,9]. N-CAM is expressed in some phases of fetal myogenesis, but is lost as development proceeds and is nearly absent from adult muscle [10]. N-CAM is expressed in satellite cells to activate and proliferate muscle bre. It is believed that N-CAM is associated with the recruitment of stem cells after muscle injury and regeneration of muscle bre [5,7,11,12,13]. After muscle bre denervation or injury, it is re-expressed again in degenerated and regenerating bres [5,7,11,12,13]. Many previous studies found that N-CAM is also presented in degenerated bres of myopathy [14] and showed a possible association between these bres in mitochondrial myopathies, in ammatory myopathies, and muscular dystrophies [14,15,16].
This study aims to explore the expression, including staining severity of N-CAM in common muscle diseases as a complement for diagnostic evaluation.

Muscle specimens
A retrospective study was performed on all muscle biopsy specimens from 2017-2019 in Ramathibodi Hospital. They included 75 clinical myopathy cases who had a muscle biopsy. The diagnosis criteria were based on both clinical correlation and histologic ndings. Clinical features and patient characteristics including age, sex, serum creatine phosphokinase level, and specimen site were obtained from the patient's medical record.

Histologic evaluation
Muscles from biceps brachii or quadriceps femoris in each case were collected as 2 cm-long specimens.
The specimens were divided into three parts. The rst part was prepared fresh for the snap-frozen section technique. The second part was performed in 10% neutral buffered formalin xation. The third part was xed in 3% glutaraldehyde for electron microscopic study.
The series of routine histochemistry stains included haematoxylin and eosin (H&E), enzymatic histochemistry, and immunohistochemistry. The muscle biopsies were evaluated and correlated with clinical information to diagnosis. (Fig. 1) In our study, we used only biopsied muscle from snap frozen section to explore the N-CAM expression. The diagnosis of muscle diseases from clinical and histopathology ndings was categorized as in ammatory myopathy, neurogenic myopathy, muscular dystrophy, mitochondrial myopathy, nonspeci c myopathy and non-diagnostic specimen.

Immunohistochemical study
The muscle biopsy specimens were snap-frozen in isopentane and liquid nitrogen. Fresh muscle specimens were rapidly frozen in isopentane (− 150 °C) and cooled in liquid nitrogen (− 80 °C). Cryostat sections (10 µm) were cut and dried on glass slides at room temperature. No xation or pretreatment was performed before the IHC analysis. Samples were incubated for 40 min with primary antibodies diluted in Bond Primary Antibody Diluent (Leica). Visualization with DAB and subsequent counterstaining with Mayer's haematoxylin were performed using the Bond Polymer Re ne Detection System (Leica). Sections were then dehydrated, cleared, and mounted. The anti-CD56 (MRQ-42) rabbit monoclonal primary antibody (1:200, Cell Marque, MilliporeSigma, Rocklin, CA, USA) was optimized for use as a fully automated IHC assay on the BenchMark ULTRA (Ventana Medical Systems Inc., Tucson, AZ). Brie y, the primary antibody was applied for 1 hour at 36 °C; ampli cation was done for 12 minutes ampli er/12 minutes multimer (OptiView Ampli cation Kit (Ventana Medical Systems Inc.), and counterstained for 16 minutes with hematoxylin II and post-counterstained with bluing reagent for 4 minutes. Palatine tonsil tissues were used as the internal control.

Interpretation
The N-CAM positivity was shown by membrane staining in regenerating bres. Generally regenerating bres were easily identi ed by enlarged nuclei with a bluish stain sarcoplasm on H&E staining, as shown in Fig. 2A. The bluish stain is due to the increased concentration of RNA within the cell [2]. Some regenerating bres looked like normal bre and were challenging to identify but showed positive N-CAM staining. The N-CAM immunohistochemical results were divided into three groups. The strongly positive had intensely diffuse membrane staining, as shown in

Statistical analysis
The relationship between N-CAM expression and common muscle diseases were evaluated using Fisher's exact versus chi-square tests. Odds ratios (ORs) were estimated in myopathies which correlated with N-CAM expression.
The p-values less than 0.05 were considered statistically signi cant and ORs more than 1.0 indicated an increased risk among the compared muscle diseases, whereas ORs less than 1.0 indicated a decrease in risk in each muscle disease. All data were analyzed by using SPSS (version 25.0.0.0).

Correlation between muscle diseases and N-CAM expression
The result of N-CAM expression showed positive staining in 35 cases and negative staining in 40 cases.
The in ammatory myopathy showed positive N-CAM in 15 out of 17 cases with statistical signi cance (pvalue < 0.001, ORs (95% CI) 14.250 (2.960-68.606)). N-CAM was also positive in muscular dystrophy, neurogenic myopathy, and nonspeci c myopathy but was not statistically signi cant in p-value. No positive N-CAM was found in mitochondrial myopathy and non-diagnostic myopathy. (Tables 1 and 2)  Table 1 Categorization of muscle disease and its correlation with N-CAM expression We also correlated the positive group with the presence of regenerating bres in histology. All positive N-CAM cases showed regenerating bres in the muscle biopsy on the haematoxylin and eosin-stained slides review. The strongly positive N-CAM mostly appeared in in ammatory myopathy and muscular dystrophy. While the weakly positive N-CAM appeared in neurogenic myopathy and nonspeci c myopathy. (Table 3) Table 3 Categorization of muscle disease and characteristics of N-CAM expression

Discussion
In this study, we showed that in ammatory myopathy was the only muscle disease that had a strong association with positive immunohistochemistry for N-CAM in muscle bres (15/17 cases, 43%, p-value < 0.01) with an odds ratio of 14.250 (95% CI = 2.960-68.606), because pathogenesis in in ammatory myopathy is either antibody/immune complex mediated or cytotoxic T-cell mediated, which results in necrosis and regeneration of muscle bres [7]. Regenerating bre which appears as a morphologically normal bre on haematoxylin and eosin stain and other histochemistry can be highlighted by immunohistochemistry against N-CAM.
We also found that only in ammatory myopathy and muscular dystrophy groups had strong-intensity immunohistochemistry against N-CAM (73% and 83%, respectively). This pattern of staining could be found in regenerating bres of muscular dystrophy, even though the overall percentage of positive N-CAM in muscular dystrophy was insigni cant (6/7 cases, 17%, p-value = 0.03).
In the mitochondrial myopathy group, our study only had 2 cases, and all cases showed negative immunohistochemistry for N-CAM. These results are limited for evaluation due to the small sample size of the specimen. The small sample size and unknown underlying pathomechanism may be the reason for the discrepancy with the previous studies.
A literature review revealed the case series of positive N-CAM in regenerating muscle bre and fetal muscle bre and negative N-CAM in normal adult muscle bre [13], and the case series of positive N-CAM in in ammatory myopathy, neurogenic myopathy and muscular dystrophy [15,16]. Nevertheless, Heuss D et al. found N-CAM positive cases in mitochondrial myopathy [14], while we found negative expression in both two mitochondrial myopathies in our study. The different results could be due to the small number of cases. (Table 4) N-CAM positive bre has been included as one of the suggestive muscle biopsy ndings for dermatomyositis according to the 239th European Neuromuscular Centre (ENMC) international workshop classi cation in 2018 [17]. (Table 5) -non-diagnostic specimen (n = 5) Table 5 The ENMC 2018 dermatomyositis classi cation criteria [17].

Conclusion
In conclusion, in ammatory myopathy showed a statistically signi cant correlation with positive N-CAM expression. The strongly positive N-CAM was found in in ammatory myopathy and muscular dystrophy. The weakly positive N-CAM was found in neurogenic myopathy and nonspeci c myopathy. No case of mitochondrial myopathy or non-diagnostic myopathy reveal N-CAM expression. All positive N-CAM cases had regenerating muscle bres on muscle biopsy on thorough review with haematoxylin and eosinstained slides. N-CAM expression accompanied by the pattern of staining could be considered to help narrow down the differential diagnosis of myopathies. We recommend further study in a larger group.    Demographic data of the patient, categorization of common muscle diseases