| Abstract|| |
Background: Fine needle aspiration cytology (FNAC) is rapid, inexpensive, and easy technique to establish the diagnosis of scalp lesions. The use of ancillary techniques such as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry on aspiration material aids in accurate diagnosis which is additionally beneficial for management and prognosis. Aims: The objective of this prospective case series was to evaluate the utility of ancillary techniques in the accurate cyto-diagnosis of malignant scalp lesions. Materials and Methods: This study was a prospective case series that included 64 cases of scalp lesions in which FNAC had been performed for diagnosis. The lesions were categorized as Non-diagnostic/Inadequate, Inflammatory, Benign and Malignant. In all the cases that were categorized as malignant additional material was collected for ancillary testing that included ICC, cell block preparation followed by IHC and flow cytometry. Results: Non-diagnostic/inadequate aspirates were 17.19% (n = 11/64), 25% (n = 16/64) aspirates were inflammatory, 35.93% (n = 23/64) aspirates were benign and 21.87% (n = 14/64) aspirates were categorized as malignant. With the aid of ancillary techniques, 57.14% malignant scalp aspirates were accurately categorized as epithelial origin. Lesions of bone and soft tissue constituted 28.57% (n = 4/14) of cases and lesions of hematolymphoid origin constituted 14.29% (n = 2/14) of all cases. Conclusion: This is a novel study where accurate categorization of malignant scalp tumors has been done with the use of ancillary techniques. This is useful as it may help in defining the tumor type, may aid in patient management. The material obtained can also be triaged for molecular testing.
Keywords: Ancillary techniques, aspiration cytology, malignant, scalp, tumors
|How to cite this article:|
Shukla S, Gupta A, Awasthi NP, Chandra S, Singh P, Kapoor S. The utility of ancillary techniques in the cyto-diagnosis of malignant scalp lesions. J Cytol 2021;38:74-81
|How to cite this URL:|
Shukla S, Gupta A, Awasthi NP, Chandra S, Singh P, Kapoor S. The utility of ancillary techniques in the cyto-diagnosis of malignant scalp lesions. J Cytol [serial online] 2021 [cited 2021 Jun 18];38:74-81. Available from: https://www.jcytol.org/text.asp?2021/38/2/74/316075
| Introduction|| |
Fine needle aspiration cytology (FNAC) plays a vital role in the diagnosis of scalp lesions as it generally avoids the hazards associated with open biopsies. It is essential to establish an accurate diagnosis pre-operatively as the management and the prognosis is dependent on accurate diagnosis. There are varied spectrums of lesions that occur in scalp that include common entities like inclusion cysts, lesions of eccrine and apocrine origin in addition to both primary and secondary malignancies., Many of the nonneoplastic entities closely mimic malignant lesions owing to the large size, gross appearance and fixity to the underlying structures. In such cases, accurate and rapid pre-operative diagnosis is essential as it defines therapeutic management. In case of malignant tumors involving the scalp rapid establishment of diagnosis through aspiration cytology will aid in rapid and targeted management. FNAC is a rapid, inexpensive and easy technique to establish the diagnosis in such cases. The objective of this prospective case series was to evaluate the utility of ancillary techniques including immunocytochemistry (ICC), immunohistochemistry (IHC) and flow cytometry in the accurate cyto-diagnosis of malignant scalp lesions.
| Materials and Methods|| |
This study was a prospective case series conducted in pathology department of a tertiary care hospital. The study included 64 cases of scalp lesions in which FNAC had been performed for diagnosis. The FNAC was performed from the lesions under aseptic conditions after written informed consent. In the case of minors, the procedure was performed only after due consent was taken from the guardian. The exact site, size, and the consistency of the swelling were documented and at least 2 passes were performed. The smears were both air dried and fixed in 95% ethyl alcohol. The staining was performed using May-Grünwald-Giemsa (MGG), hematoxylin & eosin (H&E) or Papanicolaou (Pap) stains. The lesions were categorized as Non-diagnostic/Inadequate, Inflammatory, Benign, and Malignant. In all the cases that were categorized as malignant additional material was collected for ancillary testing that included ICC, cell block preparation followed by IHC, and flow cytometry.
Preparation of cell blocks
Cell blocks were prepared by expressing the contents of the aspirated specimen onto a glass slide which allowed the specimen to dry/clot. The material was scraped off the slides and wrapped in tissue paper. The specimen was placed in a histology cassette and neutral buffered formalin was added. This was followed by processing in the automatic tissue processor using a 13-h processing schedule. The cell blocks were embedded in paraffin and sectioned at 3–4 μm thickness. The sections were stained with H&E
Protocol for ICC and IHC
ICC and IHC were performed using the standard protocol. For ICC the slides were fixed in 95% ethyl alcohol followed by antigen retrieval. For IHC performed on cell blocks tissue sections were taken on coated slides followed by de-paraffinization, rehydration, and blocking peroxidase activity. This was followed by antigen retrieval in Pascal (DAKO Cytomation, California) using sodium citrate buffer (pH-6.0). Sections were incubated for an hour with various primary antibodies [Table 1] followed by treatment with a polymer-based secondary antibody kit (Dakopatts, Envision kit, Denmark). Bound antibody was visualized using diaminobenzidine. Sections were counter-stained with hematoxylin and mounted. Positive and negative (by omitting primary antibody) controls were run with all batches.
|Table 1: List of primary IHC antibodies used for accurate characterization of malignant scalp lesions|
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Protocol for flow cytometry
Flow cytometry was performed in one of the cases suspicious for plasma cell proliferation disorder. In above case FNAC material was flushed in phosphate-buffered saline added in EDTA vial and after washing in 500 µl Phosphate buffered saline the cells were counted in Neubauer chamber to check adequacy. CD38 and CD138 were used as gating markers. Expression of CD45, CD56, CD19, CD117, Kappa, and Lambda Lights were assessed for aberrant expression on plasma cells. For assessing surface antigens, 1 x 106 cells were labeled with Fluorescent conjugated antibodies followed by lysis using BD FACS Lyse (1x) (Cat no. 349202). CD For cytoplasmic kappa and lambda light chain expression, lysis was performed by BD Pharmylse (1x) (Cat no. 555899) followed by washing and surface staining with CD38 and CD138. The cells were then fixed and permeabilized by Cytofix/cytoperm Kit (BD Cat no- 554714) followed by addition of Kappa and Lambda antibodies. After the final washing in phosphate-buffered saline cells were suspended in 1% paraformaldehyde. Sample acquisition and analyses were done on BD FACS Calibur flow cytometer (BD Biosciences) equipped with 2 laser for 4-colour immunophenotyping using Cell Quest Pro software. At least, 105 events or more were acquired in each tube. Negative limits were set by auto-fluorescence.
| Results|| |
This study was a prospective case series that included 64 cases of scalp lesion in which aspiration cytology was performed. Non-diagnostic/inadequate aspirates were 17.19% (n = 11/64), 25% (n = 16/64) aspirates were inflammatory, 35.93% (n = 23/64) aspirates were benign while 21.87% (n = 14/64) aspirates were categorized as malignant. [Table 2] and [Table 3]
|Table 3: Cytomorphological features and application of ancillary techniques in malignant scalp lesions|
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Out of the 14 malignant scalp lesions, 10 were additionally involving the underlying bone [Figure 1]a, [Figure 1]b. [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f, [Figure 1]g, [Figure 1]h, [Figure 1]i, [Figure 1]j, [Figure 1]k,[Figure 1]l. The age range of the patients varied from 6 years to 80 years with a mean age of 47.86 years. The M: F ratio was 1.3:1. Among the malignant group the lesions were further categorized based on the cell of origin as malignancies of epithelial origin, mesenchymal tumors including bone tumors and hemato-lymphoid malignancies.
|Figure 1: (a-l) Pictures of the patients showed malignant scalp lesions in first row with corresponding radiological images in second row demonstrating underlying skull involvement|
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Malignant scalp lesions of epithelial origin
In the group of malignant scalp lesion 57.14% (n = 8/14) were of epithelial origin. The age range of the cases varied from 43 years to 80 years with a mean age of 59.75 years. All the eight cases were metastatic lesions from visceral malignancies. Cytomorphologically, 7 lesions were adenocarcinomas while 1 had squamous morphology. Based on the visceral organs, the primary site was identified in the colon in 37.5% cases (n = 3/8), gall bladder in 25% cases (n = 2/8), lung in 25% cases (n = 2/8), and thyroid in 12.5% cases (n = 1/8) [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f, [Figure 2]g, [Figure 2]h.
|Figure 2: Cytosmear and cell block section showing an adenocarcinoma with corresponding cell block sections show TTF1 and napsin A positivity (a-d) consistent with primary malignancy in the lung, cytosmear and cell block section showing a papillary adenocarcinoma with corresponding cell block sections show TTF1 and thyroglobulin positivity (e-h) consistent with metastatic papillary thyroid carcinoma, cytosmear showing a malignant round cell tumor with inconspicuous vacuolated cytoplasm (i), with cytoplasmic PAS positivity (j), corresponding immunocytochemistry shows diffuse CD99 and FLI1 positivity (k,l) consistent with Ewing's sarcoma, cytosmear showing singly scattered pleomorphic epithelioid cells suggestive of pleomorphic sarcoma (m), corresponding cell block sections show diffuse vimentin, CD99 and S100 positivity (n-p) suggestive of osteosarcoma [a: MGGx400, b: H & Ex400, c,d: DABx400, e,f: H & Ex100, g: DABx100, h: DABx400, i: PAPx200, j: PASx100, k-l: DABx100, m: MGGx400, n-p: DABx400]|
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Malignant scalp lesions of bone and soft tissue (mesenchymal origin)
Lesions of bone and soft tissue constituted 28.57% (n = 4/14) of cases. All the four cases were primary tumors of scalp which included two cases of primary osteosarcoma of the skull while 2 cases were of primary ewing's sarcoma [Figure 2]i, [Figure 2]j, [Figure 2]k, [Figure 2]l, [Figure 2]m, [Figure 2]n, [Figure 2]o, [Figure 2]p. The age range varied from 6 years to 49 years with a mean age of 21.5 years. Additionally 75% (n = 3/4) cases were identified in males.
Malignant scalp lesions of hemato-lymphoid origin
Lesions of hematolymphoid origin constituted 14.29% (n = 2/14) of all cases. The mean age of the cases with hemato-lymphoid malignancies was 53 years. The first case was diagnosed as multiple myeloma with lambda chain restriction [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e. This patient had been referred from neuro-surgery outpatient department with a clinical suspicion of meningioma. The second case was reported as anaplastic large cell lymphoma-ALK negative [Figure 3]f, [Figure 3]g, [Figure 3]h, [Figure 3]i, [Figure 3]j, [Figure 3]k. This patient had clinically presented to the dermatology outpatient department with a clinical suspicion of fungal infection of the skin. Additional hematology work up in both the cases revealed systemic involvement of the hematologic malignancy.
|Figure 3: Cytosmear showing singly scattered atypical plasma cells with binucleated and multinucleated forms (a), corresponding immunocytochemistry shows diffuse vimentin, lambda light chain, CD138 and CD56 expression (b-d) consistent with neoplastic plasma cell disorder, flow cytometry on corresponding FNA material demonstrate plasma cells with CD38/138 expression, dim CD45, bright CD56 expressions, absence of CD19 expression with lambda light chain restriction, serum electrophoresis of same patient shows “M”band (e), cytosmears and cell block section showing a pleomorphic lymphoid neoplasm with singly scattered cell demonstrating doughnut/embryoid shaped nuclei and polymorphic inflammatory background (f-h), corresponding cell block sections show patchy CD45, diffuse CD3 and CD30 positivity (i-k) consistent with anaplastic large cell lymphoma [a: MGGx400, b-d: DABx200, e: flow cytometry scatter plot and serum protein electrophoresis diagram, f: MGGx400, g-h: H&Ex100, i-k: DABx400]|
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| Discussion|| |
FNAC is a rapid, accurate and inexpensive technique for the diagnosis of scalp lesions. The swellings are generally accessible and hence, additional material can be adequately retrieved for ancillary testing that further aids in diagnosis. Hingway et al., in their study have ascertained the utility of aspiration cytology in the diagnosis of scalp swellings. Accurate diagnosis is essential as further lines of management that include surgery and/or chemotherapy, radiotherapy is largely dependent on it.
In this study, 35.93% of all scalp aspirates were benign on cyto-morphology. This finding is in concordance with the studies conducted by Singh et al. and Hingway et al., wherein majority of scalp swelling were benign in nature.
Malignant scalp lesions constituted 21.87% cases in this study. This finding is supported by the results of the study conducted by Hingway et al., where malignant scalp tumors were reported in 14/80 (17.5%) cases. However, Singh et al. in their study reported malignancy in 7/185 (3.78%) aspirates.
In this study, the M: F ratio of malignant scalp tumors was 1.3:1. This finding is contradictory to the results of the studies conducted by Singh et al. and Hingway et al. who have reported a higher percentage of malignant scalp lesions in females. However, Saikia et al. have reported a higher percentage of malignant scalp tumors in males.
The scalp and the skull constitute a common metastatic site owing to the rich vasculature that serves as a fertile base for metastatic seedlings. The metastatic lesions may be present as solitary or multiple masses. Cutaneous metastasis from visceral organs constituted 57. 14% of malignant scalp lesions in this study, while Saikia et al. reported metastasis in 31.03% cases. In this study all the cases were accurately categorized with the aid of ICC and/or IHC and the primary site was accurately documented. Colon was the most common primary site followed by the gall bladder and lung. Among the various studies published in literature, this is the largest case series of somatic malignancies with scalp metastasis. In addition, the use of ancillary techniques has helped in accurate characterization of the primary site. Scalp is an easily accessible site for FNAC and accurate categorization of the primary site with the aid of ICC/IHC has an impact on treatment, as diagnostic biopsies for malignancies of visceral organs are at times technically difficult to perform and require expertise and radiological guidance. In this era of personalized medicine and targeted therapy, the material can be re-obtained from the cutaneous metastatic site for molecular marker testing. Molecular testing is now mandatory in many somatic malignancies as predictive and prognostic markers have drastic impact on survival. Testing for various markers including epidermal growth factor receptor (EGFR) mutations, ALK re-arrangements are mandatory in lung cancer. In colon carcinoma, testing for micro-satellite instability, Kirsten rat sarcoma viral oncogene homolog (KRAS) is also recommended in many cases. This is possibly the first study where such extensive IHC/ICC workup has been done to accurately categorize metastatic from visceral somatic malignancies.
Metastasis from thyroid malignancy was identified in one case (12.5%) that was morphological documented as a papillary carcinoma thyroid. This finding is contradictory to the reports published in literature by Singh et al., Saikia et al. and Hingway et al. who have reported metastasis to scalp from thyroid malignancies as the most common metastatic entity. In thyroid neoplasm, metastasis from follicular malignancies is more common and has been frequently reported. In this study, the additional novel feature was that the variant to metastasize was papillary in morphology.
Malignant scalp tumors of the hemato-lymphoid linage constituted 14.29% of all malignant aspirates. In this study, both the cases were accurately sub-categorized. The material obtained from the aspirate was utilized for FNA flow cytometry and accurate categorization was obtained. This study included one case each of multiple myeloma and anaplastic large cell lymphoma ALK-negative. Hingway et al. reported hemato-lymphoid malignancies in 4/14 (28.57%) cases. The spectrum of lesions reported by the authors included plasmacytoma, non-Hodgkin lymphoma, granulocytic sarcoma, and cutaneous T-cell lymphoma. In this study, a novel attempt was made to perform FNA flow cytometry of scalp aspirate sample to accurately categorize the hematological malignancy. This technique may prove to be cost-effective as no extra intervention will be needed to sub-categorize hematological tumors. Currently, biopsies are used for confirmatory diagnosis of lymphomas and myelomas that are generally time-consuming. The aid of ICC, IHC on cell blocks and FNA flow cytometry shall aid in rapid diagnosis and early start of treatment. This may have an impact on patient management and survival as early therapy can be initiated.,
Scalp lesions of bone and soft tissue constituted 28.57% of malignant scalp lesions. All the lesions were primary scalp tumors, which included primary osteosarcoma of the skull bone and ewing's sarcoma., In the study conducted by Hingway et al., the authors reported 2/14 (14.28%) tumors as bone or mesenchymal in origin. However, the authors were not able to accurately classify the lesions., In the current studies, all soft tissue and bone tumors were accurately categorized with the use of ancillary techniques including IHC. The accurate sub-classification of tumors further aids to triage the material for molecular testing including fluorescence in-situ hybridization for identification of translocations in ewing's tumor.
| Conclusion|| |
This is a novel study where accurate categorization of malignant scalp tumors has been done with the use of ancillary techniques including ICC, IHC, and flow cytometry. This is useful as it may help in defining the tumor type, may aid in patient management. The material obtained can also be triaged for molecular testing.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Anurag Gupta
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow – 226 010, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]