AJCP / Original Article
Expression of Bcl-xL and Mcl-1 in the Nonmelanoma Skin Cancers of Renal Transplant Recipients
CME/SAM
Michael T. Burke, MBBS,1 Christudas Morais, PhD,2 Kimberley A. Oliver, MBBS,3 Duncan L. J. Lambie, MBBS,4,5 Glenda C. Gobe, PhD,2 Robert P. Carroll, DM,6,7 Christine E. Staatz, PhD,8 Sudipta Sinnya, MBBS,9 H. Peter Soyer, MD,9 Clay Winterford, ADAB,10 Nikolas K. Haass, PhD,5 Scott B. Campbell, PhD,1 and Nicole M. Isbel, PhD1 From the Departments of 1Renal Medicine and 3Pathology, University of Queensland at Princess Alexandra Hospital, Brisbane, Australia; 2Centre for Kidney Disease Research, School of Medicine, University of Queensland, Translational Research Institute, Brisbane, Australia; 4IQ Pathology, Brisbane, Australia; 5The University of Queensland, The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia; 6Central Northern Adelaide Renal and Transplantation Services, Adelaide, Australia; 7 Department of Medicine, The University of Adelaide, Adelaide, Australia; 8School of Pharmacy, The University of Queensland, Brisbane, Australia; 9Dermatology Research Centre, The University of Queensland, School of Medicine, Translational Research Institute, Brisbane, Australia; and 10QIMR Berghofer Medical Research Institute, Brisbane, Australia. Key Words: Renal transplantation; Nonmelanoma skin cancer; Bcl-xL; Mcl-1; Immunohistochemistry; Apoptosis; Mitosis Am J Clin Pathol April 2015;143:514-526 DOI: 10.1309/AJCPQNB5WA3PLQBK
ABSTRACT Objectives: This study aims to investigate how immunosuppression influences the protein expression of antiapoptotic members of the Bcl-2 family—namely, Bcl-xL and Mcl-1—in nonmelanoma skin cancer (NMSC) and the peritumoral epidermis of renal transplant recipients. Methods: NMSC and peritumoral epidermis protein expression of Bcl-xL and Mcl-1 were assessed by immunohistochemistry in renal transplant recipients receiving tacrolimus or sirolimus and the general population not receiving immunosuppression. Results: NMSC from renal transplant recipients compared with patients not receiving immunosuppressant medications had a reduced Bcl-xL expression intensity (P = .042). Mcl-1 expression intensity in NMSC was decreased in tacrolimustreated patients compared with sirolimus-treated patients and the nonimmunosuppressed population (P = .024). Bcl-xL expression intensity was increased in peritumoral epidermis compared with NMSC (P = .002). Conclusions: It was shown for the first time that Bcl-xL and Mcl-1 expression are widespread in the peritumoral epidermis and NMSC of renal transplant recipients. Importantly in NMSC, Bcl-xL expression was reduced with immunosuppression exposure, and Mcl-1 expression was reduced in tacrolimus-treated compared with sirolimustreated patients.
514 Am J Clin Pathol 2015;143:514-526 DOI: 10.1309/AJCPQNB5WA3PLQBK
Upon completion of this activity you will be able to: • appreciate the importance of apoptosis in preventing nonmelanoma skin cancer development. • describe the mechanism by which the Bcl-2 family members Mcl-1 and Bcl-xL participate in the regulation of apoptosis. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 605. Exam is located at www.ascp.org/ajcpcme.
Renal transplantation provides the optimal long-term survival benefit for patients requiring renal replacement therapy.1 The modern era of immunosuppression has successfully reduced rates of allograft rejection and increased allograft survival, but this occurs at the cost of immunosuppressive medication-related complications.2 The risk of malignancy is increased in transplant patients and remains a significant contributor to death with a functioning renal transplant.3 Transplant recipients can develop hundreds of nonmelanoma skin cancers (NMSCs) during treatment with immunosuppressive therapies, and extensive surgery is often required. In addition, transplant recipients are more likely to develop metastatic disease,4 which is associated with increased mortality.5 The precise mechanism by which immunosuppressive medications contribute to the increased risk of NMSC is unclear but is most likely multifactorial, with medication-associated molecular effects within the epidermis an
© American Society for Clinical Pathology
AJCP / Original Article
important contributor.6 Homeostasis in the skin depends on basal keratinocyte proliferation and terminal differentiation of suprabasal keratinocytes.7 To provide an important barrier and systemic functions, epidermal keratinocytes must not only survive during differentiation but also remain susceptible to ultraviolet radiation (UVR)–induced apoptosis. UVR plays a major role in the development of NMSC through direct damage to DNA and RNA, combined with impaired anticarcinogenic mechanisms such as apoptosis and DNA repair.8 The Bcl-2 protein family is critical to the regulation of the intrinsic apoptotic pathway and the elimination of cells affected by oncogenic mutations in various human tissues, including the epidermis. The Bcl-2 family is divided into three subfamilies, including the prosurvival subfamily (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, and Bcl-B), which protects cells from apoptosis, and two additional subfamilies, many of whose members bind and inhibit the prosurvival members.9 The balance between these pro- and antiapoptotic members of the Bcl-2 family affects permeability of the outer mitochondrial membrane.9 Increased permeability causes the release of mitochondrial factors such as cytochrome C and apoptotic protease activating factor 1, which exhibit proapoptotic effects in the cytoplasm.10 Impaired apoptosis due to dysregulation of the Bcl-2 pathway is increasingly recognized as critical in the development of NMSC,7,11 along with a variety of cancers, including follicular lymphoma,12 renal cell carcinoma,13 and melanoma.14 Acquired resistance to apoptosis is a common step in the development of many cancers.9 Increased expression of Bcl-2 antiapoptotic proteins is one possible mechanism by which NMSCs arising from keratinocytes avoid apoptosis. A murine study of Bcl-xL transgenic mice showed squamous cell carcinomas (SCCs) developed earlier and were more invasive compared with those in control mice when both groups were exposed to a chemical mutagen.15 Mcl-1 is another antiapoptotic member of the Bcl-2 family that is overexpressed in a variety of cancers.16 Immunohistochemical studies show it to be prevalent throughout the adult human epidermis and to function as a major survival protein required for keratinocyte differentiation.7 In one human study, an increase in Mcl-1 expression was identified in 92% of locally advanced head and neck cancers.17 Given its essential role in the prevention of carcinogenesis, the intrinsic apoptotic pathway is a popular target for chemotherapeutic agents, and its dysregulation is one mechanism of chemoresistance.18 Mammalian target of rapamycin inhibitors (mTORi) are a commonly prescribed drug class used in renal transplantation and are unique due to their dual antineoplastic and anti– transplant rejection properties. Randomized controlled trials show a significant reduction in cutaneous SCCs in renal
© American Society for Clinical Pathology
transplant recipients (RTRs) receiving the mTORi sirolimus compared with those receiving calcineurin inhibitors.2,19,20 mTORi are proposed to reduce skin cancer through a number of mechanisms, including reduced angiogenesis,21 upregulation of T-regulatory cells,22 and cell cycle control.23 An early study linking the mTOR pathway to apoptosis and control of the cell cycle reported the mTORi rapamycininduced G1 cell cycle arrest and apoptosis in two rhabdosarcoma cell lines.24 In the skin, rapamycin in a murine model blocked UVB-induced phosphorylation of S6K, the downstream target of mTOR complex 1 (mTORC1), and significantly reduced UVB-stimulated epidermal proliferation and cell cycle progression.25 Several studies identify a relationship between the mTOR and Bcl-2 pathways. In a murine lymphoma model, constitutive expression of Mcl-1 in lymphoma cells prevented tumor regression upon mTORC1 inhibition.26 Furthermore, a study of human ovarian cancer cell lines observed resistance to rapamycin in cell lines expressing Bcl-2.27 This study aimed to identify if treatment of RTRs with immunosuppressive medications influences the expression of Bcl-xL and Mcl-1 in NMSC and the peritumoral epidermis.
Materials and Methods Patients Ethics approval for this study was obtained from the Brisbane Metropolitan South Human Research Ethics Committee. Informed consent was obtained from all patients prior to recruitment. A convenience sample of patients identified from hospital transplantation and dermatology databases were invited to participate if they had a history of NMSC excision at the Princess Alexandra Hospital, Brisbane. Demographic information on patients was collected retrospectively from patient medical records. Thirty-one NMSC and 30 peritumoral sections of epidermis from paraffin-embedded tissue blocks were examined from patients who had NMSC excisions between September 2007 and October 2013. Patients were divided into three study groups depending on immunosuppression treatment and included sirolimus-exposed RTRs (n = 10), tacrolimus-exposed RTRs (n = 11), and patients with skin cancer who had no known exposure to immunosuppressant medications (n = 10). Patients in the sirolimus and tacrolimus groups had received stable immunosuppression for greater than 6 months prior to NMSC excision. Patients were excluded from the study if they were nonwhite, had received topical imiquimod or 5-fluorouracil to the affected area, or had been given systemic chemotherapy. The excised skin lesions included
Am J Clin Pathol 2015;143:514-526 515 DOI: 10.1309/AJCPQNB5WA3PLQBK
Burke et al / Bcl-xL and Mcl-1 in Nonmelanoma Skin Cancers
intraepithelial carcinomas (IECs) and SCCs but not basal cell carcinomas (BCCs). Pathologic Study Formalin-fixed, paraffin-embedded tissue blocks containing NMSC and peritumoral epidermis were retrieved and sectioned. Specimens were deidentified with a patient code, and analyses were conducted in a blinded fashion with investigators unaware of patient groups (sirolimus, tacrolimus, or no immunosuppression). H&E-stained sections were assessed by a pathologist at the time of the initial excision and were included in the study if reported as SCC or IEC. Immunohistochemical Study Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue cut in 5-µm sections onto Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). A Leica Autostainer XL with CV5030 attachment performed immunohistochemistry and dewaxed, hematoxylin counterstained, and coverslipped the slides. Heat antigen retrieval was performed using a BioCare (Birmingham, England) medical decloaking chamber (DC2002INTL). Immunohistochemical expression was assessed with antibodies to Mcl-1 and Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA). BioCare Medical MACH 1 Probe and Polymer were applied before color development with 3,3′-diaminobenzidine. Lymphoid tissue (tonsil and spleen) served as a positive control. Appropriately diluted Jackson ImmunoResearch (West Grove, PA) ChromPure purified rabbit (Mcl-1) or mouse (Bcl-xL) IgGs were substituted for the primary antibody as negative controls. The slides were stained in a batch, ensuring comparisons in expression patterns were optimally controlled. Copies of slides were made using an APERIO XT digital pathology slide scanner (Aperio Technologies, Vista, CA) at ×20 or ×40. Two pathologists (K.A.O. and D.L.J.L.) performed the immunohistochemical analysis, and if there was disagreement, a third trained clinician (M.T.B.) determined the final result. The pathologists semiquantitatively scored the intensity of Mcl-1 and Bcl-xL protein expression (intensity) and the percentage of cells with positive Mcl-1 and Bcl-xL protein expression (expression). The intensity of immunostaining was evaluated on a scale of 0 to 3+: no staining (0), weakly positive staining (1+), moderate positive staining (2+), and strong positive staining (3+) ❚Image 1❚. The percentage of positive cells was estimated and categorized as 0% to 33%, 34% to 66%, and 67% to 100%. The subcellular location of Mcl-1 and Bcl-xL protein expression (nuclear, cytoplasmic, or membranous) was recorded. In 30 of the 31 specimens, immunohistochemical expression was compared between NMSC and areas of peritumoral epidermis. One NMSC did not have an area of adjacent epidermis to compare.
516 Am J Clin Pathol 2015;143:514-526 DOI: 10.1309/AJCPQNB5WA3PLQBK
Apoptosis and Mitosis Apoptosis was reported using the in situ enzymatic assay ApopTag (ApopTag Peroxidase in situ Apoptosis Detection Kit, Merck-Millipore, Billerica, MA) and Aperio digital images at ×40 in association with specific morphologic features that have been described previously.28 Mitotic cells were identified according to morphologic features and counted using H&E-stained slides at ×400.29 Cell counting was performed by a single trained investigator (M.T.B.). Apoptotic and mitotic cells were counted within four randomly selected grids for NMSC and peritumoral epidermis. Apoptotic and mitotic cells were expressed as a mean percentage of the total number of cells within the grids. Statistical Analysis Statistical analysis was performed with Stata version 12 (StataCorp LP, College Station, TX). Quantitative data were assessed for normality. Patients’ demographic characteristics were summarized using median and interquartile range (IQR) for quantitative data and counts (percentage) for categorical data. Mean differences in NMSC and peritumoral Mcl-1 and Bcl-xL protein expression were compared between groups using a Fisher exact test. Mean differences in apoptosis and mitosis between groups were compared using a Kruskal-Wallis test. A P value of
Expression of Bcl-xL and Mcl-1 in the Nonmelanoma Skin Cancers of Renal Transplant Recipients
CME/SAM
Michael T. Burke, MBBS,1 Christudas Morais, PhD,2 Kimberley A. Oliver, MBBS,3 Duncan L. J. Lambie, MBBS,4,5 Glenda C. Gobe, PhD,2 Robert P. Carroll, DM,6,7 Christine E. Staatz, PhD,8 Sudipta Sinnya, MBBS,9 H. Peter Soyer, MD,9 Clay Winterford, ADAB,10 Nikolas K. Haass, PhD,5 Scott B. Campbell, PhD,1 and Nicole M. Isbel, PhD1 From the Departments of 1Renal Medicine and 3Pathology, University of Queensland at Princess Alexandra Hospital, Brisbane, Australia; 2Centre for Kidney Disease Research, School of Medicine, University of Queensland, Translational Research Institute, Brisbane, Australia; 4IQ Pathology, Brisbane, Australia; 5The University of Queensland, The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia; 6Central Northern Adelaide Renal and Transplantation Services, Adelaide, Australia; 7 Department of Medicine, The University of Adelaide, Adelaide, Australia; 8School of Pharmacy, The University of Queensland, Brisbane, Australia; 9Dermatology Research Centre, The University of Queensland, School of Medicine, Translational Research Institute, Brisbane, Australia; and 10QIMR Berghofer Medical Research Institute, Brisbane, Australia. Key Words: Renal transplantation; Nonmelanoma skin cancer; Bcl-xL; Mcl-1; Immunohistochemistry; Apoptosis; Mitosis Am J Clin Pathol April 2015;143:514-526 DOI: 10.1309/AJCPQNB5WA3PLQBK
ABSTRACT Objectives: This study aims to investigate how immunosuppression influences the protein expression of antiapoptotic members of the Bcl-2 family—namely, Bcl-xL and Mcl-1—in nonmelanoma skin cancer (NMSC) and the peritumoral epidermis of renal transplant recipients. Methods: NMSC and peritumoral epidermis protein expression of Bcl-xL and Mcl-1 were assessed by immunohistochemistry in renal transplant recipients receiving tacrolimus or sirolimus and the general population not receiving immunosuppression. Results: NMSC from renal transplant recipients compared with patients not receiving immunosuppressant medications had a reduced Bcl-xL expression intensity (P = .042). Mcl-1 expression intensity in NMSC was decreased in tacrolimustreated patients compared with sirolimus-treated patients and the nonimmunosuppressed population (P = .024). Bcl-xL expression intensity was increased in peritumoral epidermis compared with NMSC (P = .002). Conclusions: It was shown for the first time that Bcl-xL and Mcl-1 expression are widespread in the peritumoral epidermis and NMSC of renal transplant recipients. Importantly in NMSC, Bcl-xL expression was reduced with immunosuppression exposure, and Mcl-1 expression was reduced in tacrolimus-treated compared with sirolimustreated patients.
514 Am J Clin Pathol 2015;143:514-526 DOI: 10.1309/AJCPQNB5WA3PLQBK
Upon completion of this activity you will be able to: • appreciate the importance of apoptosis in preventing nonmelanoma skin cancer development. • describe the mechanism by which the Bcl-2 family members Mcl-1 and Bcl-xL participate in the regulation of apoptosis. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 605. Exam is located at www.ascp.org/ajcpcme.
Renal transplantation provides the optimal long-term survival benefit for patients requiring renal replacement therapy.1 The modern era of immunosuppression has successfully reduced rates of allograft rejection and increased allograft survival, but this occurs at the cost of immunosuppressive medication-related complications.2 The risk of malignancy is increased in transplant patients and remains a significant contributor to death with a functioning renal transplant.3 Transplant recipients can develop hundreds of nonmelanoma skin cancers (NMSCs) during treatment with immunosuppressive therapies, and extensive surgery is often required. In addition, transplant recipients are more likely to develop metastatic disease,4 which is associated with increased mortality.5 The precise mechanism by which immunosuppressive medications contribute to the increased risk of NMSC is unclear but is most likely multifactorial, with medication-associated molecular effects within the epidermis an
© American Society for Clinical Pathology
AJCP / Original Article
important contributor.6 Homeostasis in the skin depends on basal keratinocyte proliferation and terminal differentiation of suprabasal keratinocytes.7 To provide an important barrier and systemic functions, epidermal keratinocytes must not only survive during differentiation but also remain susceptible to ultraviolet radiation (UVR)–induced apoptosis. UVR plays a major role in the development of NMSC through direct damage to DNA and RNA, combined with impaired anticarcinogenic mechanisms such as apoptosis and DNA repair.8 The Bcl-2 protein family is critical to the regulation of the intrinsic apoptotic pathway and the elimination of cells affected by oncogenic mutations in various human tissues, including the epidermis. The Bcl-2 family is divided into three subfamilies, including the prosurvival subfamily (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, and Bcl-B), which protects cells from apoptosis, and two additional subfamilies, many of whose members bind and inhibit the prosurvival members.9 The balance between these pro- and antiapoptotic members of the Bcl-2 family affects permeability of the outer mitochondrial membrane.9 Increased permeability causes the release of mitochondrial factors such as cytochrome C and apoptotic protease activating factor 1, which exhibit proapoptotic effects in the cytoplasm.10 Impaired apoptosis due to dysregulation of the Bcl-2 pathway is increasingly recognized as critical in the development of NMSC,7,11 along with a variety of cancers, including follicular lymphoma,12 renal cell carcinoma,13 and melanoma.14 Acquired resistance to apoptosis is a common step in the development of many cancers.9 Increased expression of Bcl-2 antiapoptotic proteins is one possible mechanism by which NMSCs arising from keratinocytes avoid apoptosis. A murine study of Bcl-xL transgenic mice showed squamous cell carcinomas (SCCs) developed earlier and were more invasive compared with those in control mice when both groups were exposed to a chemical mutagen.15 Mcl-1 is another antiapoptotic member of the Bcl-2 family that is overexpressed in a variety of cancers.16 Immunohistochemical studies show it to be prevalent throughout the adult human epidermis and to function as a major survival protein required for keratinocyte differentiation.7 In one human study, an increase in Mcl-1 expression was identified in 92% of locally advanced head and neck cancers.17 Given its essential role in the prevention of carcinogenesis, the intrinsic apoptotic pathway is a popular target for chemotherapeutic agents, and its dysregulation is one mechanism of chemoresistance.18 Mammalian target of rapamycin inhibitors (mTORi) are a commonly prescribed drug class used in renal transplantation and are unique due to their dual antineoplastic and anti– transplant rejection properties. Randomized controlled trials show a significant reduction in cutaneous SCCs in renal
© American Society for Clinical Pathology
transplant recipients (RTRs) receiving the mTORi sirolimus compared with those receiving calcineurin inhibitors.2,19,20 mTORi are proposed to reduce skin cancer through a number of mechanisms, including reduced angiogenesis,21 upregulation of T-regulatory cells,22 and cell cycle control.23 An early study linking the mTOR pathway to apoptosis and control of the cell cycle reported the mTORi rapamycininduced G1 cell cycle arrest and apoptosis in two rhabdosarcoma cell lines.24 In the skin, rapamycin in a murine model blocked UVB-induced phosphorylation of S6K, the downstream target of mTOR complex 1 (mTORC1), and significantly reduced UVB-stimulated epidermal proliferation and cell cycle progression.25 Several studies identify a relationship between the mTOR and Bcl-2 pathways. In a murine lymphoma model, constitutive expression of Mcl-1 in lymphoma cells prevented tumor regression upon mTORC1 inhibition.26 Furthermore, a study of human ovarian cancer cell lines observed resistance to rapamycin in cell lines expressing Bcl-2.27 This study aimed to identify if treatment of RTRs with immunosuppressive medications influences the expression of Bcl-xL and Mcl-1 in NMSC and the peritumoral epidermis.
Materials and Methods Patients Ethics approval for this study was obtained from the Brisbane Metropolitan South Human Research Ethics Committee. Informed consent was obtained from all patients prior to recruitment. A convenience sample of patients identified from hospital transplantation and dermatology databases were invited to participate if they had a history of NMSC excision at the Princess Alexandra Hospital, Brisbane. Demographic information on patients was collected retrospectively from patient medical records. Thirty-one NMSC and 30 peritumoral sections of epidermis from paraffin-embedded tissue blocks were examined from patients who had NMSC excisions between September 2007 and October 2013. Patients were divided into three study groups depending on immunosuppression treatment and included sirolimus-exposed RTRs (n = 10), tacrolimus-exposed RTRs (n = 11), and patients with skin cancer who had no known exposure to immunosuppressant medications (n = 10). Patients in the sirolimus and tacrolimus groups had received stable immunosuppression for greater than 6 months prior to NMSC excision. Patients were excluded from the study if they were nonwhite, had received topical imiquimod or 5-fluorouracil to the affected area, or had been given systemic chemotherapy. The excised skin lesions included
Am J Clin Pathol 2015;143:514-526 515 DOI: 10.1309/AJCPQNB5WA3PLQBK
Burke et al / Bcl-xL and Mcl-1 in Nonmelanoma Skin Cancers
intraepithelial carcinomas (IECs) and SCCs but not basal cell carcinomas (BCCs). Pathologic Study Formalin-fixed, paraffin-embedded tissue blocks containing NMSC and peritumoral epidermis were retrieved and sectioned. Specimens were deidentified with a patient code, and analyses were conducted in a blinded fashion with investigators unaware of patient groups (sirolimus, tacrolimus, or no immunosuppression). H&E-stained sections were assessed by a pathologist at the time of the initial excision and were included in the study if reported as SCC or IEC. Immunohistochemical Study Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue cut in 5-µm sections onto Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). A Leica Autostainer XL with CV5030 attachment performed immunohistochemistry and dewaxed, hematoxylin counterstained, and coverslipped the slides. Heat antigen retrieval was performed using a BioCare (Birmingham, England) medical decloaking chamber (DC2002INTL). Immunohistochemical expression was assessed with antibodies to Mcl-1 and Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA). BioCare Medical MACH 1 Probe and Polymer were applied before color development with 3,3′-diaminobenzidine. Lymphoid tissue (tonsil and spleen) served as a positive control. Appropriately diluted Jackson ImmunoResearch (West Grove, PA) ChromPure purified rabbit (Mcl-1) or mouse (Bcl-xL) IgGs were substituted for the primary antibody as negative controls. The slides were stained in a batch, ensuring comparisons in expression patterns were optimally controlled. Copies of slides were made using an APERIO XT digital pathology slide scanner (Aperio Technologies, Vista, CA) at ×20 or ×40. Two pathologists (K.A.O. and D.L.J.L.) performed the immunohistochemical analysis, and if there was disagreement, a third trained clinician (M.T.B.) determined the final result. The pathologists semiquantitatively scored the intensity of Mcl-1 and Bcl-xL protein expression (intensity) and the percentage of cells with positive Mcl-1 and Bcl-xL protein expression (expression). The intensity of immunostaining was evaluated on a scale of 0 to 3+: no staining (0), weakly positive staining (1+), moderate positive staining (2+), and strong positive staining (3+) ❚Image 1❚. The percentage of positive cells was estimated and categorized as 0% to 33%, 34% to 66%, and 67% to 100%. The subcellular location of Mcl-1 and Bcl-xL protein expression (nuclear, cytoplasmic, or membranous) was recorded. In 30 of the 31 specimens, immunohistochemical expression was compared between NMSC and areas of peritumoral epidermis. One NMSC did not have an area of adjacent epidermis to compare.
516 Am J Clin Pathol 2015;143:514-526 DOI: 10.1309/AJCPQNB5WA3PLQBK
Apoptosis and Mitosis Apoptosis was reported using the in situ enzymatic assay ApopTag (ApopTag Peroxidase in situ Apoptosis Detection Kit, Merck-Millipore, Billerica, MA) and Aperio digital images at ×40 in association with specific morphologic features that have been described previously.28 Mitotic cells were identified according to morphologic features and counted using H&E-stained slides at ×400.29 Cell counting was performed by a single trained investigator (M.T.B.). Apoptotic and mitotic cells were counted within four randomly selected grids for NMSC and peritumoral epidermis. Apoptotic and mitotic cells were expressed as a mean percentage of the total number of cells within the grids. Statistical Analysis Statistical analysis was performed with Stata version 12 (StataCorp LP, College Station, TX). Quantitative data were assessed for normality. Patients’ demographic characteristics were summarized using median and interquartile range (IQR) for quantitative data and counts (percentage) for categorical data. Mean differences in NMSC and peritumoral Mcl-1 and Bcl-xL protein expression were compared between groups using a Fisher exact test. Mean differences in apoptosis and mitosis between groups were compared using a Kruskal-Wallis test. A P value of
