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Automated 5 Minute Mohs Micrographic Surgery Immunohistochemistry
Authors: Alfonso Heras, DVM, PhD¹, Tahnia Mark¹, Yanyan Lu, PhD¹, Preyas Shah ²
Institutions: 1. Bio SB, Inc. Santa Barbara, CA, [email protected]
2. Rushabh Instruments, LLC, Warrington, PA Purpose: The most common reason for recurrence of skin cancers after Mohs micrographic surgery (MMS) is residual undetected tumor. Frozen tissue immunohistochemistry (IHC) has demonstrated greater sensitivity than routine H&E stains, which are difficult to interpret. We report the use of a highly sensitive monovalent Fab anti‐mouse and anti‐rabbit micropolymer IHC detection system for the detection of IVD rabbit and mouse monoclonal antibodies for melanoma (Melanoma cocktail <HMB45+MART‐1+Tyrosinase>, MART‐1, SOX‐10, and S‐100), BCC and SCC (CK AE1/AE3 and EpCAM BerEP4) using a compact, easy to program and easy to operate 5‐slide (simultaneously or independently) automated IHC stainer. The Automated Mohs IHC Stainer unit is configured to work with a 7 reagent cartridge and 7 dispense positions containing all the reagents and solutions necessary to conduct the IHC. Each slide holder can be easily and independently programmed for time, temperature, reagent application and rinses. Design: A total of 14 cases (5 melanomas, 4 BCC and 5 SCC) frozen MMS biopsies were serially cut at 4‐5 um, mounted on Hydrophilic Plus slides and fixed for 2 min. in 100% acetone. The remaining frozen tissues were formalin‐fixed, paraffin‐embedded (FFPE) and used as a gold standard control. IHC and H&E results were blindly evaluated in each section, by qualified personnel. Statistical analysis was performed using a T‐test to determine equivalency or statistically significant differences of the different antibodies, detection HRP & AP systems, incubation times, substrate‐chromogens and frozen vs FFPE results of the Mohs IHC. The following was the 5‐min. or 10‐min. IHC protocol used for both Frozen and FFPE tissues (subjected to prior deparaffinization and HIER) using the 5‐slide Automated Mohs IHC Stainer:
Step 5 Min. Protocol 10 Min. Protocol
Peroxidase Block 1 min. 2 min.
Antibody 1 min. 2 min.
Anti‐Mouse or Rabbit Fab Micropolymer HRP or AP 1 min. 2 min.
Brown, Red, Green, Magenta or Blue Chromogen 1 min. 2 min.
Counterstain 0.5 min. 1.5 min.
Washes between steps 0.5 min. 0.5 min.
Results: No statistically significant differences (p>0.05) were observed in the tumor cell density and signal distribution in the 5 or 10 min. Mohs IHC for all the IHC markers tested on frozen sections compared to the FFPE paraffin sections for HRP DAB, HRP Green and HRP Blue. Both HRP/AEC and AP/ALK Scarlet had statistically significant differences with less sensitivity on both frozen and FFPE tissues, when compared to HRP DAB, HRP Green and HRP Blue. The automated 5 or 10 min. Mohs IHC in frozen and paraffin sections
correlated with the H&E staining of normal and tumoral cells in paraffin sections with no statistically significant differences for all the experimental antibodies. Conclusion: Automated 5 Mohs IHC stain is a reproducible, specific, sensitive and reliable adjunctive diagnostic method to aid in the interpretation of surgical margins during MMS for melanoma, BCC and SCC. The technique is superior to frozen or permanent H&E sections alone, and is equivalent to the IHC of the same FFPE tissue sections. The automated protocol allows fast and reproducible IHC staining with minimal labor, which increases the precision and efficiency of the Mohs procedure.
INTRODUCTION
Complete tumor excision of skin cancers depends on the diagnostic accuracy in the evaluation of surgical margins during Mohs micrographic surgery (MMS). The most common reason for recurrence of tumor after Mohs micrographic surgery is residual undetected tumor. Because hematoxylin and eosin stains may present difficulties in interpretation, immunohistochemistry techniques are being used to supplement these routine stains. Although immunohistochemistry is not being widely utilized by Mohs micrographic surgery surgeons, the many advantages of immunohistochemistry over routine staining of frozen sections in selected settings is of great value (1).
There have been different attempts to improve IHC for MMS. Cherpelis et al, reported the development of an effective ultra‐rapid cytokeratin (CK) frozen section 19‐minute immunostain to be used during MMS in cases of non‐melanoma skin cancer (NMSC) with dense or perineural inflammation and concluded that this innovative 19‐minute ultra‐rapid CK immunostain can be used to detect trace quantities of NMSC in frozen sections during MMS (2). Chang et al, conducted a study to determine if an automated 16 minute protocol for MART‐1 stain was a reliable tool during MMS for melanoma and concluded that automated MART‐1 IHC stain is a rapid and reliable adjunctive diagnostic method to aid in the interpretation of surgical margins during MMS for melanoma (3). Valentin et al, studied 2,122 cutaneous melanomas (CM) excised by MMS and frozen section examination of margins using MART‐1 immunostain and concluded that MMS with MART‐1 is an effective treatment modality for CM while decreasing the uncertainty of H&E frozen section interpretation with the additional advantage of easier interpretation (4). The introduction of highly validated IVD non‐biotin polymer IHC detection systems (5), new sensitive mouse and rabbit monoclonal antibodies, substrate‐chromogen and automated IHC systems, have opened the doors for a faster and more accurate immunohistochemistry applicable to MMS. We report the use of a highly sensitive monovalent Fab anti‐mouse and anti‐rabbit micropolymer IHC detection system for the detection of IVD monoclonal antibodies for Melanoma, BCC and SCC using a compact, easy to program and operate 5‐slide (operated simultaneously or independently of other slides) automated IHC stainer.
DESIGN
1. TISSUE SPECIMENS 1.1. A total of 14 cases (5 melanomas, 4 BCC and 5 SCC) frozen MMS biopsies were serially cut at 4‐5 um,
mounted on Hydrophilic Plus slides and fixed for 2 min in 100% acetone. The remaining frozen tissues were formalin‐fixed, paraffin‐embedded (FFPE) and used as a gold standard control.
1.2. Formalin‐fixed paraffin‐embedded 23‐core Normal Human Tissue Microarrays (NH TMA) and 11‐core Cancer Human TMA (CA TMA) serially cut at 4‐5 um, mounted on Hydrophilic Plus slides containing Melanoma, BCC and SCC were used as controls.
FIGURE 1. Tissue Maps of the 23‐core Normal Human TMA and 11‐core Cancer Human TMA used for the Different Automated Mohs
Immunohistochemistry Studies
23‐Core Normal Human Tissue Microarray (NH TMA)
11‐core Cancer Human TMA (Ca TMA)
Placenta Blank Breast Uterus Cervix Fallopian T.
Brain Pituitary Adrenal Pancreas Salivary Colon
Liver Kidney Thyroid Lung Skin Bladder
Testis Prostate Spleen Tonsil Bone M. Thymus
NHL Blank Ovarian Ca Testicular Ca
Colon Ca SCC TCC Breast Ca
Prostate Ca Thyroid Ca Melanoma Ovarian Ca
2. ANTIBODIES 2.1. Antibodies used to test the different Frozen or FFPE Tissues by IHC are considered in Table 1.
TABLE 1. Antibodies used to test the different Frozen or FFPE Tissues with by the Automated Mohs IHC Stainer System
Antibody Clone Isotype Expected Signal
Frozen Tissues
FFPE Control Tissues
Melanoma
Cocktail
HMB‐45
A103
BSB‐6
IgG1/K
IgG1
IgG2a
Cytoplasmic Melanoma NH TMA
CA TMA
MART‐1 A103 IgG1 Cytoplasmic Melanoma NH TMA
CA TMA
SOX‐10 BSB‐62 IgG2b/K Nuclear Melanoma NH TMA
CA TMA
S‐100 4C4.9 IgG2a Cytoplasmic
Nuclear
Melanoma NH TMA
CA TMA
CK AE1/AE3 AE1/AE3 IgG1 Cytoplasmic BCC
SCC
NH TMA
CA TMA
EpCAM BerEP4 Ber‐EP4 IgG1/K Cytoplasmic BCC
SCC
NH TMA
CA TMA
3. DETECTION SYSTEMS
The following was the 5‐ or 10‐min. IHC protocol used for both frozen and FFPE tissues (subjected to prior deparaffinization and Heat‐induced Epitope Retrieval (HIER*)) using the 5‐slide automated IHC stainer:
TABLE 2. Detection System Steps used to test the different Frozen or FFPE Tissues
using the Automated Mohs IHC Stainer System
Step 5 Min. Protocol 10 Min. ProtocolPeroxidase Block 1 min. 2 min.
Antibody 1 min. 2 min.
Anti‐Mouse or Rabbit Fab Micropolymer HRP or AP 1 min. 2 min.
Brown, Red, Green, Magenta or Blue Chromogen 1 min. 2 min.
Counterstain 0.5 min. 1.5 min.
Washes between steps 0.5 min. 0.5 min.
*FFPE Tissues were deparaffinized and subjected to Citrate pretreatment for 15 min. at 121 C using a pressure cooker.
FIGURE 2. Mouse or Rabbit Fab’ MicroPolymer HRP or AP Detection System Graphical Depiction and Steps used to test the different Frozen or FFPE Tissues
with the Automated Mohs IHC Stainer System
4. EQUIPMENT
FIGURE 3. Automated Mohs IHC Stainer Prototype
The Automated Mohs IHC Stainer is a compact, easy to program and operate 5‐slide (simultaneously or independently) automated IHC stainer. The Automated Mohs IHC Stainer unit is configured to work with a 7 reagent cartridge and 7 dispense positions containing all the reagents and solutions necessary to conduct the IHC. Each slide holder can be easily and independently programmed for time, temperature, reagent application and rinses. No pipetting is needed, plus hazardous and non‐hazardous waste are collected separately.
5. ACCEPTANCE CRITERIA
5.1. The IHC specific signals and background were scored by qualified professionals using a scale of 0 to 4. For Frozen and FFPE TMA’s the signal had to be cell compartment specific and tissue specific. Tissue cores from the TMA’s that are known to lack the antigen for a specific antibody, had to be negative.
5.2. Results were evaluated using a paired T‐test to determine equivalency or statistically significant
differences of the different antibodies, detection HRP & AP systems, incubation times, substrate‐chromogens and frozen vs FFPE results of the Fast MOHS IHC.
RESULTS
TABLE 3. Average IHC Signal and Background of Different Antibodies with 5 min. vs 10 min. Fab Micropolymer Detection System HRP/DAB using FFPE Tissues with
the Automated Mohs IHC Stainer System
Antibody 5 Min. Protocol 10 Min. ProtocolMelanoma 3.40/0.25 3.60/0.25
MART‐1 3.50/0.25 3.60/0.25
SOX‐10 3.25/0.25 3.50/0.25
S‐100 3.25/0.25 3.50/0.25
CK AE1/AE3 3.50/0.25 3.50/0.25
EpCAM Ber‐EP4 3.25/0.25 3.50/0.25
No statistically significant differences were observed between the 5 min. vs the 10 min. MOHS IHC protocol for all the experimental antibodies. The 10 min. Mohs IHC protocol produced stronger signals than the 5 min. protocol and was especially evident with SOX‐10, S‐100 and EpCAM Ber‐EP4.
TABLE 4. Average IHC Signal and Background of Different Antibodies with a 5 min. Fab Micropolymer HRP or AP Detection System with Different Substrate‐
Chromogens on Frozen and FFPE Tissues using the Automated Mohs IHC Stainer System
FROZEN Mohs IHC
FFPE IHC
Antibody HRP/DAB HRP/AEC HRP/Green HRP/Blue AP/ALK Scarlet HRP/DAB
Melanoma 3.50/0.25 3.25/0.25 3.50/0.25 3.50/0.25 3.00/0.25 3.50/0.25
MART‐1 3.50/0.25 3.00/0.25 3.50/0.25 3.25/0.25 3.00/0.25 3.50/0.25
SOX‐10 3.25/0.25 2.75/0.25 3.25/0.25 3.37/0.25 2.75/0.25 3.25/0.25
S‐100 3.25/0.25 2.75/0.25 3.25/0.25 3.37/0.25 3.00/0.25 3.25/0.25
CK AE1/AE3 3.50/0.25 3.00/0.25 3.50/0.25 3.50/0.25 3.00/0.25 3.50/0.25
EpCAM Ber‐EP4 3.25/0.25 3.00/0.25 3.25/0.25 3.25/0.25 2.75/0.25 3.25/0.25
No statistically significant differences (p>0.05) were observed in the tumor cell density and signal distribution in the 5 min. or 10 min. Mohs IHC for all the IHC markers tested on frozen sections compared to the FFPE paraffin sections for HRP DAB, HRP Green and HRP Blue. Both HRP/AEC and AP/ALK Scarlet had statistically significant differences with less sensitivity on both frozen and FFPE tissues, when compared to HRP DAB, HRP Green and HRP Blue.
The automated 5 min. or 10 min. Mohs IHC in frozen and paraffin sections correlated with the H&E staining of normal and tumoral cells in paraffin sections with no statistically significant differences for all the experimental antibodies.
FIGURE 4. IHC of MART‐1 on Melanoma and CK AE1/AE3 on SCC Frozen Sections using the Fab Micropolymer HRP DAB, HRP Green or ALK Scarlet and the
Automated Mohs IHC Stainer System
Automated 5 min. Mohs IHC HRP DAB of MART‐1 on
a Frozen Melanoma
Automated 5 min. Mohs IHC HRP Green of MART‐1 on a Frozen Melanoma
Automated 10 min. Mohs IHC AP ALK Scarlet of MART‐1 on a Frozen
Melanoma
Automated 5 min. Mohs IHC HRP DAB of CK AE1/AE3
on a Frozen SCC
Automated 5 min. Mohs IHC HRP Green of CK
AE1/AE3 on a Frozen SCC
Automated 10 min. Mohs IHC ALK Scarlet of CK
AE1/AE3 on a Frozen SCC
CONCLUSIONS
The automated Mohs IHC Stainer System is a fast, reproducible, specific, sensitive and reliable adjunctive diagnostic method to aid in the interpretation of surgical margins during MMS for melanoma, BCC and SCC. The technique is superior to frozen or permanent H&E sections alone, and is equivalent to the IHC of the same FFPE tissue sections. The automated protocol allows fast and reproducible IHC staining with minimal labor, which increases the precision and efficiency of the Mohs IHC procedure.
REFERENCES
1. Sroa, N, et al. Immunohistochemistry in Mohs Micrographic Surgery: A Review of the Literature. J Clin Aesthetic Dermatol. 2009; 2(7):37–42.
2. Cherpelis, BS, at al. Innovative 19 Minute Rapid Cytokeratin Immunostaining of Non‐melanoma Skin Cancer
in Mohs Micrographic Surgery. 41st Mohs College Annual Meeting. Austin, TX. April, 2009. 3. Chang, KH, et al. An Automated 16‐Minute Technique for Processing Mohs Sections for Melanoma. 41st
Mohs College Annual Meeting. Austin, TX. April, 2009. 4. Valentin, SM, et al. The Utility of MART‐1 Immunostains in the Management of Invasive Melanoma and
Melanoma In Situ with Mohs Micrographic Surgery. 45th Mohs College Annual Meeting. Washington, DC, May, 2013.
5. Heras, A, et al. Enhanced Polymer Detection System for Immunohistochemistry. Lab Invest 1995; 72:165.