{'fraction_non_alphanumeric': 0.021641719897034988, 'fraction_numerical': 0.009343121365239775, 'mean_word_length': 5.560350218886804, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Vimentin and Cytokeratin Immunostaining: The Role in Basic Diagnosis and Prognosis of Sarcomas

Introduction: Specific diagnosis which provides diagnostic, prognostic, and therapeutic information to guide patient care defines the primary goal of sarcoma management and care. The role of immunohistochemistry, using vimentin (mesenchymal linage marker) and cytokeratin (epithelial linage marker) as basic markers for diagnosis and classification of sarcomas for specific management strategies and prognosis was elucidated in the present study. Materials and Methods: Twenty four (24) archived paraffin wax processed tissue block sarcoma samples were randomly selected from the histopathology Laboratories and museums of the Nnamdi Azikiwe University Teaching Hospital (NAUTH) Nnewi and National hospital Abuja and necessary data obtained from records. Blocks were re-embedded with fresh paraffin wax and 3μ thick sections cut with the aid of a rotary microtome. Haematoxylin and Eosin staining method was employed to confirm diagnosis before proceeding to immunohistochemistry. Antibodies for vimentin and pancytokeratin were employed for immunohistochemistry while exposed mouse and rabbit specific horseradish peroxides/diaminobenzidine detection IHC kit was employed for immunostaining. Results: The mean age of patients was 26 years while the ages range from 11 to 48 years with 14 (58.3%) females and 10 (41.7%) males . Vimentin had strong positive immunoreactivity for all sarcoma samples whereas cytokeratin had positive immunoreactions for synovial sarcoma only, which also showed co-expression of both genes. Conclusion: Vimentin and cytokeratin may play vital role as basic biomarkers not only for diagnosis and characterization of sarcomas but for specific management regime and prognostication. However, IHC must be performed at high standard using appropriate antibodies, samples and


INTRODUCTION
Vimentin is a gene which encodes for type III intermediate filament protein. Intermediate filaments, along with microtubules and actin microfilaments constitute the cytoskeleton. It has a role in neuritogenesis and cholesterol transport and functions as an organizer of a number of other critical proteins involved in cell attachment, migration, and signaling with its mutations associated with congenital cataracts in human patients. Vimentin expressed in certain types of carcinomas (renal cell carcinoma, spindle cell carcinoma), as well as lymphomas and melanomas It is also is expressed in mesenchymal cells though, not specific for them 1 . Due the crucial role of vimentin in maintaining muscle cyto-architecture, it is considered an essential marker for muscle regeneration 2 . Cytokeratins are members of the keratin gene family and consists of basic or neutral proteins arranged in pairs of heterotypic keratin chains and co-expressed during differentiation of simple and stratified epithelial tissues. According to Wei  The authors further noted frequent co-expression of vimentin and cytokeratin in some carcinomas which may suggest certain types of epithelial tumors as possible primary sites in the evaluation of metastatic tumors.
Sarcomas are connective tissues malignant neoplasms usually found in the skeletal muscle, fat, tendons, fibrous tissue, smooth muscle and the neurovascular elements that support these components and in bone with diverse and varied histogenesis 6 . Though, believed to account for 1% of all human malignancies, the growing incidence, the very aggressive and invasive nature and the propensity of most sarcomas to metastasize has brought it to limelight as a major health challenge 6 . Sarcomas have predilections for children and young adults with a male to female ratio of 2:1 7,8 . Dermatofibrosarcoma protuberans, malignant peripheral nerve sheath tumour, osteoblastic osteosarcoma, fibromyxoma, malignant mesenchymal tumour, fibromyxoma, alveolar rhabdomyosarcoma, metastatic liposarcoma, synovial sarcoma, low grade leiomyosarcoma to mention but a few are examples of most commonly diagnosed sarcoma cases in Nigeria 8 . The wide heterogeneous nature of sarcomas, with greater than 100 histological types and subtypes with considerable morphological overlaps between the different diagnostic entities 5 makes it very challenging in establishing definitive diagnosis of most sarcoma types. Furthermore, sarcomas assume poorly differentiated, undifferentiated with no obvious line of differentiation or may even form as secondary implant (metastatic tumours) in distant sites, thereby increasing the challenge of definitive diagnosis. According to Wei et al 1 the general approach to reaching a definitive differential diagnosis of soft tissue tumors is to first consider clinicoradiologic, histomorphologic, and cytomorphologic features of the tumour. Tumours with obvious line of differentiation such as smooth-muscle, skeletal-muscle, vascular, neural, and Chondro-osseous lineages may be diagnosed with morphological features. Contrariwise however, immunostaining, using appropriate marker will be required to actualize diagnosis.
In order actualize definitive and specific diagnosis that provides diagnostic, prognostic, and therapeutic information to guide patient care, immunohistochemistry play a crucial role 1 . The role of immunohistochemical expressions of vimentin and cytokeratin, as afore stated, in the basic classification of sarcomas cannot be overemphasized. The present study aimed at evaluating the immunohistochemical expression pattern of vimentin and cytokeratin using selected samples of commonly diagnosed sarcomas in our facility. The expression pattern among the implications will highlight the role of the genes in basic characterization of sarcomas which is a basic a critical step towards patients' care and management especially in a middle income country like Nigeria.

Study area/ Study design
This was a cross sectional study using 24 archived samples of already diagnosed sarcoma which was carried out at the Nnamdi Azikiwe University Teaching Hospital, Nnewi and National hospital Abuja.

Ethical approval
Ethical approval to carry out this study was obtained from the Ethical Committee of Nnamdi Azikiwe University Teaching Hospital (NAUTH) Nnewi.

Sample collection
Twenty four (24) archived paraffin wax processed tissue block sarcoma samples were randomly selected from the histopathology Laboratories and museums of the Nnamdi Azikiwe University Teaching Hospital (NAUTH) Nnewi and National hospital Abuja. Necessary data were obtained from clinical records, operation notes and histopathology reports of the patients.

Tissue preparation
Tissue blocks were re-embedded in fresh paraffin wax, 3µ thick sections cut with the aid of a rotary microtome, cut sections floated out on a lukewarm Leica water bath, mounted on slides previously coated with poly-l-lysine, drained, labelled and placed on Leica hot plate in order to dry and affix the tissue onto the slides.

Staining
Cut Sections were stained by Haematoxylin and Eosin (H&E) method and morphological diagnosis of each sample confirmed before proceeding to immunostaining.

Immunohistochemical Staining (IHC)
IHC of test materials and positive controls were carried according to a method by Nishio et al 9 . Antibodies for vimentin and pancytokeratin were employed for immunohistochemistry. Exposed Mouse and Rabbit Specific horseradish peroxidase/diaminobenzidine (HRP/DAB) detection IHC kit was employed for immunostaining while detection of immunoreactivity was performed according to manufacturer's instruction. Both antibodies and detection kits were procured from Abcam Plc Cambridge UK.

Immunoreactivity Scoring
Immunoreactivity was semi-quantitatively scored according to Zlobec et al 10 . This was based on percentage of cells (area) that will stain positive and the intensity of the staining (strong, moderate, weak). A score of 5+ was assigned to 80% or more of epithelial and/ or stromal cells that stained positive with strong intensity, 4+ was assigned to 50% or more (but less than 80%) of epithelial cells and/ stromal cells with strong intensity or 80% of cells or more with moderate to weak intensity; 3+ was assigned to 30% or more of epithelial and/ or stromal cells with strong intensity or 50% or more (but less than 80%) of positive cells with moderate to weak intensity; 2+ was assigned to 10% or more cells that stained positive with strong intensity or 30% or more ( but less than 50%) that stained moderate to weak and 1+ was assigned to 10% or more cells ( less than 30% of positive cells) that stained positive with moderate to weak intensity; 0 was assigned to less than 10% of positivity irrespective of the intensity of staining.

Data Analyses
Numerical data were summarized using mean and standard deviation, whereas categorical data were being presented using frequency and proportion. Immunoreactivity pattern was expressed as percentages.

RESULTS
The mean age of patients was 26 years while the ages range from 11 to 48 years with 14 (58.3%) females and 10 (41.7%) males. The breakdown of sarcoma samples were as follows: 4 dermatofibrosarcoma protuberans, 4 malignant peripheral nerve sheath tumour, 4 fibromyxoma and 2 each of osteoblastic osteosarcoma, malignant mesenchymal tumour, alveolar rhabdomyosarcoma, metastatic liposarcoma, synovial sarcoma and low grade leiomyosarcoma. Histopathological and immunoreactivity features for vimentin and cytokeratin were as shown in Table 1. Vimentin had strong positive immunoreactivity for all sarcoma samples where as cytokeratin had positive immunoreactions for synovial sarcoma only, which also showed co-expression of both genes Photomicrographs of immunohistochemical staining pattern of anti vementin and cytokeratin sarcoma samples are as shown in (Figure 1).     Parham 5 in his reported on the imperative of IHC and its potential applications, not just for diagnosis and prognostication, but for personalized therapy decisions. The strong positivity of vimentin in all sarcoma samples studied not only agreed with previous studies but validates its presence in mesenchymal tumours.

CONCLUSION
It could be concluded from the finding of the present study that vimentin and cytokeratin may play vital role as basic biomarkers not only for diagnosis and characterization of sarcomas but for specific management regime and prognostication. However, IHC must be performed at high standard using appropriate antibodies, samples and reagents.