Histopathologic characteristics of therapy-associated cutaneous neoplasms with vemurafenib, a selective BRAF kinase inhibitor, used in the treatment of melanoma.

BACKGROUND: Activating mutations in BRAF have been observed in up to 60% of melanomas, indicating a pivotal role for kinase deregulation in tumor progression. Vemurafenib is a specific inhibitor of BRAF for treatment of melanomas with activating BRAF V600E mutations and has been a major advancement in melanoma treatment. Treatment with vemurafenib, and to a lesser extent, sorafenib, a relatively non-specific inhibitor of BRAF, has been associated with cutaneous squamous cell carcinoma (SCC). METHODS: Clinical and microscopic characteristics of cutaneous neoplasms were evaluated following vemurafenib administration. RESULTS: Twenty-four of 47 (51%) patients receiving vemurafenib at our institution developed 146 total cutaneous neoplasms, with 75% developing multiple lesions. The median number of lesions in affected patients was three. Body distribution included head/neck (29%), chest/back (21%), upper (23%) and lower extremities (27%). Lesions were biopsied and pathologically showed multiple types of epidermal tumors including, but not limited to, verrucous keratoses with/without partial thickness dysplasia, actinic keratoses and well-differentiated and invasive SCCs with/without keratoacanthomatous features. CONCLUSIONS: We describe the histopathologic findings of skin lesions potentially associated with vemurafenib. Additional investigation is necessary to further elucidate cutaneous neoplasms associated with vemurafenib; however, frequent dermatologic evaluation is warranted in all patients receiving BRAF inhibitors.

Considerations on the performance of immunohistochemistry for mismatch repair gene proteins in cases of sebaceous neoplasms and keratoacanthomas with reference to Muir-Torre syndrome.

Cutaneous sebaceous tumors, as well as keratoacanthomas, are associated with Muir-Torre syndrome (MTS). Visceral neoplasias are a feature of this syndrome; thus, early detection using a cutaneous biopsy is very important in dermatopathology. A dermatopathologist can apply different techniques in such cases, including immunohistochemistry for several mismatch repair proteins or molecular studies for microsatellites instability. However, in a world where economic resources play an increasing role, how discriminative the dermatopathologist should be in investigating cutaneous lesions that may indicate MTS is not a trivial matter. Although previous reports have presented algorithms on MTS focusing on a multidisciplinary approach, our report emphasizes the role of the dermatopathologist and tries to answer several questions that could arise when facing a cutaneous tumor that may indicate MTS.

Treatment of multiple keratoacanthomas with erlotinib.

An 82-year-old male presented with numerous pruritic erythematous nodules over his trunk and extremities. Histopathology was consistent with keratoacanthomas. Given the extent of his disease, medical therapy was recommended. Based on phosphorylated epidermal growth factor receptor expression in lesional keratinocytes, treatment with erlotinib 150 mg daily was initiated, with rapid improvement in the number and appearance of nodules. Immunohistochemistry following treatment revealed a decrease in lesional pEGFR expression, consistent with inhibition of this receptor activation. This is the first report of multiple keratoacanthomas responding to therapy with an EGFR tyrosine kinase inhibitor, and it supports an emerging role for the use of EGFR inhibitors in the management of cutaneous malignancies.

Ligand activation of peroxisome proliferator-activated receptor-beta/delta and inhibition of cyclooxygenase-2 enhances inhibition of skin tumorigenesis.

Ligand activation of peroxisome proliferator-activated receptor (PPAR)-beta/delta and inhibition of cyclooxygenase-2 (COX-2) activity by nonsteroidal anti-inflammatory drugs can attenuate skin tumorigenesis. There is also evidence that attenuation of skin tumorigenesis by inhibition of COX-2 activity occurs through PPARbeta/delta-independent mechanisms. The present study examined the hypothesis that combining ligand activation of PPARbeta/delta with inhibition of COX-2 activity will cooperatively inhibit chemically induced skin tumor progression using both in vivo and ex vivo models. A two-stage chemical carcinogenesis bioassay was performed in wild-type and Pparbeta/delta-null mice. After 22 weeks, cohorts of both mouse lines were divided into four experimental groups: (1) control, (2) topical application of the PPARbeta/delta ligand GW0742, (3) dietary administration of the COX-2 inhibitor nimesulide, or (4) both GW0742 and nimesulide. Ligand activation of PPARbeta/delta did not influence skin tumor progression, while a modest decrease in skin tumor multiplicity was observed with dietary nimesulide. Interestingly, the combined treatment of GW0742 and nimesulide increased the efficacy of the decrease in papilloma multiplicity for 6 weeks in wild-type mice, but this effect was not found at later time points and was not found in similarly treated Pparbeta/delta-null mice. Neoplastic keratinocyte lines cultured with GW0742 and nimesulide also exhibited enhanced inhibition of cell proliferation coincident with increased expression of Keratin messenger RNAs. Results from these studies support the hypothesis that combining ligand activation of PPARbeta/delta with inhibition of COX-2 activity can inhibit chemically induced skin tumor progression by modulating differentiation.

PGP 9.5 expression in cutaneous keratoacanthomas and squamous cell carcinomas.

The aim of the study was to investigate the expression of PGP 9.5 in cutaneous keratoacanthomas (KAs) and squamous cell carcinomas (SCCs). Thirty-one cases of KA (10 in the growth stage, 9 in the mature phase and 12 in the involution stage) and 36 SCCs including 13 well differentiated cases, 12 moderately differentiated tumors, 7 poorly differentiated lesions and 4 pseudoadenoid entities were investigated. PGP 9.5 expression was positively correlated with tumor stage (P < 0.001) and potential perineural invasion (P < 0.001). There was no significant difference in the distribution of patients presenting variable levels of PGP 9.5 staining with regard to maximal tumor size and the extent and degree of stromal invasion. PGP 9.5 expression proved closely associated with tumor aggressiveness and is classified as a marker for predicting the outcome of resection-treated skin cancer patients.

Expression of apoptotic and cell proliferation regulatory proteins in keratoacanthomas and squamous cell carcinomas of the skin.

The aim of this study was to investigate the expression of p53, caspase-3, bcl-2, MIB-1, and PCNA to validate more objective methods to differentiate squamous cell carcinoma and keratoacanthoma, as well as to understand their pathogenesis with accuracy. A total of 52 cases of histopathologically diagnosed keratoacanthoma in the proliferative stage and 56 cases of well-differentiated squamous cell carcinoma were selected in this study. The expression was evaluated by means of immunohistochemistry. Bcl-2 immunoreactivity was weak or absent in the majority of cases, either in squamous cell carcinoma or in keratoacanthoma. PCNA-positive cells did not show differences between two lesions evaluated. On the other hand, MIB-1 was statistically significant (p<0.05) between squamous cell carcinomas and keratoacanthomas. Moreover, p53 and caspase-3 were overexpressed in squamous cell carcinomas. Together, these results suggest that the biological behavior of the well-differentiated squamous cell carcinomas of the skin may be associated with cellular proliferation and/or deregulation of cell death, indicated by increased expression of the MIB-1 and apoptotic proteins p53 and caspase-3, respectively.

The multi-step process of human skin carcinogenesis: a role for p53, cyclin D1, hTERT, p16, and TSP-1.

As proposed by Hanahan and Weinberg (2000. Cell 100, 57-70) carcinogenesis requires crucial events such as (i) genomic instability, (ii) cell cycle deregulation, (iii) induction of a telomere length maintenance mechanism, and (iv) an angiogenic switch. By comparing the expression of p53, cyclin D1, p16, hTERT, and TSP-1 in spontaneously regressing keratoacanthoma (KA) as a paradigm of early neoplasia, with malignant invasive cutaneous squamous cell carcinoma (SCC) as a paradigm of advanced tumour development, we are now able to assign the changes in the expression of these proteins to specific stages and allocate them to defined roles in the multi-step process of skin carcinogenesis. We show that mutational inactivation of the p53 gene, and with that the onset of genomic instability is the earliest event. Individual p53-positive cells are already seen in "normal" skin, and 3/5 actinic keratoses (AKs), 5/22 KAs, and 13/23 SCCs contain p53-positive patches. Cell cycle deregulation was indicated by the overexpression of the cell cycle regulator cyclin D1, as well as by the loss of the cell cycle inhibitor p16. Interestingly, overexpression of cyclin D1 - observed in 80% of KAs and SCCs, respectively - showed a cell cycle-independent function in HaCaT cell transplants on nude mice. Cyclin D1 overexpression was associated with a massive inflammatory response, finally leading to tissue destruction. Loss of the cell cycle inhibitor p16, on the other hand, correlated with SCCs. Thus, it is tempting to suggest that overexpression of cyclin D1 is an early change that in addition to growth stimulation leads to an altered epithelial-mesenchymal interaction, while functional p16 is able to control this deregulated growth and needs to be eliminated for malignant progression. Another requirement for uncontrolled growth is the inhibition of telomere erosion by up-regulating telomerase activity. As measured by hTERT protein expression, all of the KAs and SCCs studied were positive, with a similar distribution of the protein in both groups and an expression pattern resembling that of normal epidermis. Thus, telomerase may not need to be increased significantly in skin carcinomas. Finally, we show that the angiogenesis inhibitor TSP-1 is strongly expressed in most KAs, and mainly by the tumour cells, while in SCCs the generally weak expression is restricted to the tumour-stroma. Furthermore, we provide evidence that the loss of a copy of chromosome 15 is responsible for reduced TSP-1 expression and thereby this aberration contributes to tumour vascularisation (i.e. the angiogenic switch) required for malignant growth.

Transformation-specific matrix metalloproteinases, MMP-7 and MMP-13, are present in epithelial cells of keratoacanthomas.

Keratoacanthomas are rapidly growing hyperproliferative skin tumors that may clinically or histologically be difficult to distinguish from well-differentiated squamous cell cancers (SCCs). UV light, trauma, and immune suppression represent their etiological factors. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated the expression profile of several cancer-related MMPs to find markers that would differentiate keratoacanthomas from SCCs and shed light to the pathobiology of keratoacanthoma. Samples from 31 keratoacanthomas and 15 grade I SCCs were studied using immunohistochemistry for MMP-2, -7, -8, -9, -10, -13, and -19 and p16 and laminin-5gamma2 chain. In situ hybridization for MMP-7, -10, and -13 was performed in a subset of tumors. Keratinocytes with atypia, presence of neovascularization, and composition of the inflammatory infiltrate were graded from hematoxylin-eosin stainings. MMP-7 was present in the epithelium of 4/31 keratoacanthomas and 9/15 SCCs, MMP-8 in 3/30 keratoacanthomas and 0/15 SCCs, but MMP-13 in 16/31 keratoacanthomas and 10/15 SCCs, and MMP-10 in 28/31 keratoacanthomas and all cancers. MMP-9 was detected in the epithelium in 5/31 keratoacanthomas and 8/15 SCCs, whereas MMP-2 was only present in fibroblasts in both tumors. MMP-19 was upregulated in proliferating epithelium of keratoacanthomas as was p16. Cytoplasmic laminin-5gamma2 was particularly abundant in keratinocytes at the pushing border of MMP-13-positive keratoacanthomas. We conclude that although some MMPs (MMP-10 and -13) are abundantly expressed in keratoacanthomas, the presence of MMP-7 and -9 in their epithelial pushing border is rare and should raise suspicion of SCC. Further, the loss of MMP-19 and p16 could aid in making the differential diagnosis between well-differentiated SCC and keratoacanthoma. Frequent expression of the transformation-specific MMP-13 in keratoacanthomas suggests that they are not benign tumors but incomplete SCCs.

Biological behavior of keratoacanthoma and squamous cell carcinoma: telomerase activity and COX-2 as potential markers.

Distinguishing keratoacanthoma from squamous cell carcinoma is a persistent issue in pathology practice. Solitary keratoacanthoma is a self-limiting lesion as opposed to rather aggressive clinical behavior of squamous cell carcinoma. Several markers were studied to understand their biology and to separate these two lesions on a firm basis, but without much success. In this study, we plan to utilize recent markers such as telomerase activity and cyclooxygenase-2 (COX-2) along with more established marker p53 in understanding the biologic differences between keratoacanthoma and squamous cell carcinoma. We studied 17 well to moderately differentiated squamous cell carcinoma and 24 early proliferative phase keratoacanthoma by immunohistochemistry for the expression of p53 protein, COX-2 and telomerase activity. Higher telomerase activity was found in 11/17 squamous cell carcinoma (65%) compared to 4/24 (17%) of keratoacanthoma. Similarly, stronger expression of p53 and COX-2 was detected in 12 (71%) and 11 (65%) cases of squamous cell carcinoma compared to 2 (8%) and 2 (8%) cases of keratoacanthoma respectively. A highly significant 'P' value was obtained for telomerase activity (0.001), p53 (0.000), and COX-2 (0.001). Telomerase activity, COX-2, and p53 expression provide evidence that keratoacanthoma and squamous cell carcinoma are indeed distinct entities and also help in discriminating these two lesions, which closely resemble each other on conventional morphology. Although these markers present new insights into the biologic variation of keratoacanthoma and squamous cell carcinoma, they are of limited value for routine application in histological distinction of these two lesions. The differential expression of markers also explains the sustained proliferation observed in squamous cell carcinoma, compared to a shorter lifespan and involution in keratoacanthoma.

Expression of the cyclin-dependent kinase inhibitor p27 in keratoacanthoma.

BACKGROUND: Keratoacanthomas are characterized by initial rapid enlargement followed by clinical regression. A series of cyclin and cyclin-dependent kinase complexes regulate cell cycle progression. p27(kip) inhibits a variety of cyclin-cyclin-dependent kinase complexes in vitro and may act to hold eukaryotic cells in a quiescent state (G0). OBJECTIVE: We examined expanding and regressing keratoacanthomas for expression of p27(kip). METHODS: An immunohistochemical method was used to visualize and count p27(kip)-labeled cells in 5 expanding and 15 regressing keratoacanthomas. RESULTS: In normal epidermis p27(kip) was found overlying the nuclei of suprabasilar keratinocytes. In expanding keratoacanthoma there was little expression of p27(kip) in nuclei of atypical keratinocytes composing the tumor (1.25 +/- 2.1 labeled cells per high-power field); in regressing keratoacanthoma the nuclei of most suprabasilar keratinocytes in atypical tumor aggregates contained p27(kip) (55.1 +/- 28.6 labeled cells per high-power field). The difference was significant at P values of less than.001. CONCLUSION: The identification of p27(kip) in regressing keratoacanthoma but not in expanding keratoacanthoma suggests that p27(kip) may be playing a role in promoting regression of keratoacanthoma and is a potential target for pharmacologic intervention.

Expression of stromelysin 3 in keratoarcanthoma and squamous cell carcinoma.

Stromelysin 3 (ST3) is a member of the metalloproteinase family, which is expressed in the skin during wound healing and in the stroma of basal cell carcinoma. A high level of expression of ST3 has been observed in carcinomas of poor prognosis. In benign tumors, though, ST3 is not expressed or is at a low level. We have immunohistochemically studied the expression of ST3 in 89 randomly selected cases of squamous cell carcinomas (SCCs), 104 of keratoacanthomas (KA), and 23 cases of metastatic SCC. Stromelysin 3 was expressed only by fibroblasts surrounding the tumors and not by epithelial cells. The proportion of tumors positively stained was 22% of KA, 47% of randomly selected SCC, and 70% of metastatic SCC. In areas of poorly demarcated neoplastic cells, a reinforcement of the staining was observed in the stroma. The intensity and dispersion of staining were used to determine the level of expression. There were significantly more SCC in the groups of high expression levels, and both parameters were significantly higher in SCC than in KA. Expression of ST3 in benign tumors is unusual. Its expression in the the stroma of keratoacanthomas can be related to the high tissue remodeling activity observed in these tumors. It also could be interpreted as in favor of the neoplastic nature of KA. Nevertheless, the level of expression was higher in SCC than in KA and seemed to be related to the prognosis of these tumors. These results correlate well with those obtained in breast cancers and in noncutaneous SCC.

Immunolocalizations of human gelatinase (type IV collagenase, MMP-9) and TIMP (tissue inhibitor of metalloproteinases) in normal epidermis and some epidermal tumors.

The matrix metalloproteinases (MMPs) MMP-2 and MMP-9 (gelatinases) have been suggested as serving an important role in cleaving the basement membrane structure. Tissue inhibitors of metalloproteinases TIMPs (particularly TIMP-1) are known to inhibit MMPs. Based on this background, we raised monoclonal antibodies against human gelatinase (MMP-9) and human recombinant TIMP (TIMP-1), and immunostained these two components in skin from patients with squamous cell carcinoma (SCC), Bowen's disease (BD) and keratoacanthoma (KA). MMP-9 showed positive staining mainly in the granular layer of normal epidermis. In some cases of SCC and BD, MMP-9 showed positive staining in the dysplastic lesions even in the basal layer. TIMP showed a thorough positivity in normal epidermis. Unstained regions with this antibody were observed in SCC and BD. These results suggest that an altered staining pattern for MMP-9 and TIMP may be closely related to the malignant transformation of SCC and BD.

Expression of the nucleoside diphosphate kinase in human skin cancers: an immunohistochemical study.

Expression of nucleoside diphosphate(NDP) kinase, which is homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential. However, the relationship remains controversial according to the tumor cell types and experimental system, with conflicting results from different research groups. In order to determine whether NDP kinase expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of NDP kinase expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of NDP kinase was intense in KA and SCC compared with BCC. However, the difference of NDP kinase expression between KA and SCC was not statistically significant. And there was no statistically significant difference in NDP kinase expression between SCC with metastasis and SCC without metastasis. Our results contradict the hypothesis concerning the possible role of nm23 gene as a metastatic suppressor gene in human skin cancer. The mechanism of overexpression in various tumor cell types and its biological significance in cutaneous carcinogenesis remain to be determined.

Expression of the nucleoside diphosphate kinase in human skin cancers: an immunohistochemical study

Expression of nucleoside diphosphate(NDP) kinase, which is homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential. However, the relationship remains controversial according to the tumor cell types and experimental system, with conflicting results from different research groups. In order to determine whether NDP kinase expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of NDP kinase expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of NDP kinase was intense in KA and SCC compared with BCC. However, the difference of NDP kinase expression between KA and SCC was not statistically significant. And there was no statistically significant difference in NDP kinase expression between SCC with metastasis and SCC without metastasis. Our results contradict the hypothesis concerning the possible role of nm23 gene as a metastatic suppressor gene in human skin cancer. The mechanism of overexpression in various tumor cell types and its biological significance in cutaneous carcinogenesis remain to be determined.

Enzymes of carbohydrate metabolism in human epidermal tumors. 5.

The activities of eight enzymes were determined by fluorometric micromethods in epidermal tumors (basal and squamous cell epithelioma, verruca seborroeica and keratoacanthoma) and compared to the activities in the adjoining, apparently normal epidermis. The activities were increased in basal cell epithelioma, variable in squamous cell epithelioma and verruca seborroeica and lowered in keratoacanthoma.

Enzymes of carbohydrate metabolism in human epidermal tumors 4. Enzymes of the pentose phosphate pathway.

The acitvities of six enzymes were determined by fluorometric micromethods in epidermal tumors (basal and squamous cell epithelioma, verruca seborrhecia and keratoacanthoma) and compared to the activities in the adjoining, apparently normal epidermis. Specific changes related to malignancy were not obvious.

Enzymes of carbohydrate metabolism in human epidermal tumors. 3. Enzymes between glucose and triose-phosphate.

The activities of five enzymes were determined by fluorometric micromethods in epidermal tumors (basal and squamous cell epithelioma, verruca seborrhoica and keratocanthoma) and compared to the activities in the adjoining, apparently normal epidermis. Most activities were higher in the tumoral tissues.