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Immunohistochemical Staining in the Mohs Lab

By: Jon Hetts, BS, HT (ASCP)


The application of Immunohistochemistry (IHC) staining in the Mohs lab can allow for improved treatment of melanoma and non-melanoma skin cancers. Mohs micrographic surgery (MMS) has largely been a preferred treatment for non-melanoma skin cancers. [1] Traditional hematoxylin and esoin staining techniques made diagnosis of melanoma very difficult. [1] The development of IHC stains allowed for more accurate diagnosis in routine pathology yet these new approaches were not easily applied in Mohs. Advances in IHC detection kits and antibody clonality has opened the door for integration with the Mohs lab.


Mohs is a microscopically controlled surgical procedure in which thin layers of tissue are removed and prepared for frozen sectioning. The stained frozen sections are evaluated in stages until the surgical margins are clear of cancer cells. This technique allows for minimal loss of healthy tissue, minimal scarring, improved recovery time, and improved cure rate [2]. Recurrence of skin cancer after Mohs surgery is largely due to residual undetected cancer cells. [1]


The gold standard for histopathologic evaluation has always been the hematoxylin and eosin (H&E) stain. Detection and evaluation of the relative distribution of melanocytes, the cells that produce melanin, is critical in diagnosing melanoma. The H&E stain makes it hard to distinguish between melanocytes and other surrounding cells in the basal layer of the epidermis. In the H&E stain of MMS frozen tissue in image 1, the melanocyte (arrow) is hard to distinguish from other surrounding basal cells of the epidermis. Due to the limitations of the H&E stain immunohistochemical stains have been developed to aid in the histopathologic interpretation. [1]


Image 1: H&E frozen section stain of skin showing melanocyte (arrow).

There are a few IHC antibodies that can specifically bind to melanocytes and visually highlight just these cells, making it much easier to evaluate melanocyte populations. The melanoma antigen recognized by T-cells 1 (MART-1) is a commonly used IHC stain in routine Pathology and when used in the Mohs lab in conjunction with the H&E stain, can make diagnosis and treatment of malignant melanoma (MM) and melanoma in situ (MIS) more successful. [1] Image 2 shows the same tissue from image 1 stained with a rapid 14 minute MART-1 IHC stain protocol, melanocytes are clearly highlighted in brown. The standard chromogen for IHC is diaminobenzidine (DAB), a brown enzyme mediated colored product.


Because melanocytes produce melanin, a brown pigment, it is sometimes helpful to use another color chromogen such as purple or red. In image 3, a purple chromogen (Bajoran Purple from Biocare) is used in place of DAB allowing for easy delineation of melanocytes from the brown melanin pigment. Sox-10 is another useful immunostain for the evaluation of melanoma margins. Whereas MART-1 is a cytoplasmic stain that binds to melanocytes, Sox-10 is a nuclear stain. The frozen Mohs tissue in image 4 shows the nuclear staining of melanocytes using Sox-10 on a rapid 25 minute protocol.


Image 2: MART-1 frozen section IHC stain of skin highlighting just melanocytes in brown (arrow).

Image 3: MART-1 with purple chromogen highlighting melanocytes around melanin pigment.

Image 4: Sox10 with purple chromogen highlighting melanocytes on frozen Mohs tissue.

The application of Immunohistochemical (IHC) stains for hard to diagnose cases in routine pathology is standard practice and has been used along side the H&E stain for over a decade. [4] IHC staining in the Mohs lab has not been a commonly utilized staining technique largely due to time constraints of traditional IHC staining protocols which were developed for formalin fixed paraffin embedded samples. [1] Mohs relies upon a rapid (4-10 minute) H&E stain for diagnosis and maintaining a similar turn around time is critical if IHC staining is to be adopted in the Mohs lab.


Immunohistochemical staining is a multi-step staining process based upon antigenicity, affinity of a primary antibody (antigen) to bind to a specific protein (epitope) of a cell. [5]Traditionally, IHC staining on formalin fixed paraffin embedded (FFPE) tissue was a lengthy process that could take hours to perform, making it relatively impractical in the Mohs lab. Much of the time needed for IHC staining on FFPE tissue involves epitope retrieval. During formalin fixation proteins and peptides are cross-linked, “masking” a primary antibodies specific protein binding site (epitope). In order to perform IHC stains on FFPE tissue you need to “unmask” these protein crosslinks, a process called epitope retrieval. [5]


Unlike FFPE tissue, Mohs tissue is freshly frozen and not formalin fixed or paraffin embedded. This gives Mohs tissue the ability to proceed with IHC staining protocols without epitope retrieval. Additionally freshly frozen tissue preserves the tissues antigenicity; affinity for a primary antibody to bind to a target epitope. This characteristic can allow for shorter incubation times on each IHC protocol step. Depending on the type of IHC detection kit you use there can be anywhere from 2-5 steps in your IHC staining protocol.


Advancements in micro polymer detection kits and antibody clones has allowed for rapid IHC staining protocols. [1] Using higher antibody concentrations can also reduce incubation times leading to a reduced protocol time. [1] Using the latest in rapid IHC detection kits some frozen tissue protocols can be a short as 5 minutes.


Image 5: CK7 stain on frozen Mohs tissue highlighting dermal involvement of Paget cells.

The new rapid IHC staining kits and improved antibody clones have removed many of the time constraining barriers that once prevented application of IHC stains in the Mohs lab. The utility of IHC in MMS is not limited to Melanoma, but can be useful in the treatment of Extramammary Paget’s Disease and Dermatofibrosarcoma Protuberans (DFSP). Cytokeratin 7 (CK7) is a preferred immunostain for the evaluation of margins for Extramammary Paget’s Disease. [1] Image 5 shows MMS tissue from a Extramammary Paget’s case staining with CK7 using a rapid 14 minute IHC stain protocol. The CD34 antibody is a common cell marker for the evaluation of DFSP margins. [1] Image 6 shows MMS tissue from a DFSP case stained with CD34 using a rapid 14 minute IHC stain protocol.


Image 6: CD34 stain on frozen Mohs tissue.

Want to learn more about IHC in the Mohs lab? Check out Jonathon's workshop at the NSH Symposium/Convention in New Orleans this September.


References:

1. Novie Sroa, MD, Shannon Campbell, DO, and Larisa Ravitskiy, MD. J Clin Aesthnet Dermatol. 2009 July 2(7):37-42. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924140/

2. Sheila Valentin-Nogueras, MD, FAAD, David Brodland, MD, FAAD, FACMS, John Zitelli, MD, FAAD, FACMS, Lorena Gonzalez-Sepulveda, MS, and Cruz Nazario, PhD. Dermatol Surg. 2016 June: 42(6):733-744. Retrieved from https://www.ncbi.nlm.nih.gov/pm/c/articles/PMC5422990

3. Albertini JG, Elston DM, Libow LF, Smith SB, Farley MF. Dermatol Surg. 2002 Aug, 28(8):656-65. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/12174054

4. Fan Lin and Zongming Chen (2014) Standardization of Diagnostic Immunohistochemistry: Literature Review and Geisinger Experience. Archives of Pathology & Laboratory Medicine: December 2014, Vol. 138, No. 12, pp. 1564-1577. Retrieved from https://www.archivesofpathology.org/doi/full/10.5858/arpa.2014-0074-RA

5. Russell Myers, PhD. Technical Brief of Heath Induced Epitope Retrieval. Retrieved from https://www.leicabiosystems.com/pathologyleaders/technical-brief-of-heat-induced-epitope-retrieval/

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