Engineering a waste management enzyme to overcome cancer resistance to apoptosis: adding DNase1 to the anti-cancer toolbox.

Cancer treatment is often complicated by resistance to conventional anti-cancer treatment and to more recently developed immunotherapy and gene therapy. These therapeutic modalities aim at activating death pathways within cancer cells. Attempts to activate the apoptotic death pathway, by overexpressing proapoptotic signals, are compromised by cancer defense mechanisms, which disrupt the apoptotic-signaling cascade downstream of the overexpressed component. Here, we describe a therapeutic option of triggering apoptosis without activating the apoptotic-signaling cascade or using the native apoptosis executioner nuclease. We have engineered Deoxyribonuclease-1 (DNase1), a waste-management enzyme, by deleting its signal peptide, adding a nuclear localization signal, and mutating its actin-binding site. Apoptosis studies and colony-forming assay for assessing cell viability were conducted in apoptosis-resistant Mel-Juso human melanoma cells. The modified DNase1 reduced cell viability by 77% relative to controls. It also induced typical microscopic features of cellular apoptosis, such as Terminal Transferase dUTP Nick-End Labeling-positive cells and DNA fragmentation. Quantification of apoptosis by Laser scanning cytometry demonstrated high-killing efficiency of 70-100%. The results suggest that this modified DNase1 can efficiently eliminate apoptosis-resistant cancer cells through apoptosis. Coupled to different tissue-specific gene expression elements, this recombinant DNase1 may serve as a platform for eliminating a variety of cancer types.

[Desmoplastic Spitz nevus: a histopathological review and comparison with desmoplastic melanoma]. / Naevus de Spitz desmoplasique : analyse histopathologique et comparative avec le mélanome desmoplasique.

INTRODUCTION: Desmoplastic Spitz nevus is a rare variant of spitzoid tumours characterized by dermal proliferation of large epitheliod and/or fusiform melanocytes within a desmoplastic stroma, comprising thick, eosinophilic collagen bundles. It occurs most frequently in young adults and children, and exhibits a predilection for the limbs. Histologically, this entity may be mistaken for cutaneous fibrous tumours or desmoplastic melanoma. To establish useful histological criteria for differential diagnosis of desmoplastic Spitz nevus, we conducted a retrospective study comparing data concerning the clinical, demographic and histological characteristics of desmoplastic Spitz nevus and desmoplastic melanoma. PATIENTS AND METHODS: We reviewed the slides for 28 cases of desmoplastic Spitz nevus and for 13 cases of desmoplastic melanoma obtained from the files of Pinkus Dermatopathology Laboratory (Monroe, MI, USA) during the period 1993-2005. RESULTS: Desmoplastic Spitz nevus was more common in women (sex ratio M/F=0.4) and younger patients (mean age: 28.2 years) whereas the mean age of patients with desmoplastic melanoma was significantly higher (mean age: 75 years). From a histopathological point of view, symmetry, circumscription, melanocytic maturation and involvement of adnexal structure were significantly more frequent in desmoplastic Spitz nevi. The presence of melanocytic junctional nests associated with discohesive cells, variations in size and shape of the nests, lentiginous melanocytic proliferation, actinic elastosis, pagetoid spread, dermal mitosis, perineural involvement and brisk inflammatory infiltrate were significantly more frequent in desmoplastic melanoma. No significant difference was found concerning epidermal hyperplasia, presence of Kamino bodies or moderate inflammatory infiltrate. CONCLUSION: The combination of architectural and cytological features was useful in differentiating desmoplastic Spitz nevus from desmoplastic melanoma. A complete excision biopsy is mandatory in such tumors as the architectural criteria are necessary for a correct diagnosis.

Utility of anti-bacillus Calmette-Guérin antibodies as a screen for organisms in sporotrichoid infections.

BACKGROUND: Sporotrichoid infections often present a diagnostic challenge for the dermatopathologist. Therefore an affordable, reliable method of easier recognition of organisms is desired. Recently, immunohistochemical staining with antibodies against Mycobacterium bovis (bacillus Calmette-Guérin, BCG) was found to be useful in identifying various fungi and mycobacteria with minimal background staining. OBJECTIVE: The purpose of this study was to demonstrate the usefulness of anti-BCG antibodies as a screen for organisms in sporotrichoid infections. METHODS: Thirteen specimens of suspected sporotrichosis were selected for staining with anti-BCG antibody. Sporotrichoid infection was confirmed by histochemical staining, biopsy, and follow-up results. RESULTS: Twelve of the 13 specimens stained positively using anti-BCG antibody. Of the 5 cultures done, 2 were positive for M. marinum, and 1 grew Sporothrix schenckii. CONCLUSION: Immunohistochemical staining with anti-BCG antibody offers another screening method to identify organisms in sporotrichoid infections because of its ease, cost-effectiveness, and simplicity.

A study of apoptosis in Merkel cell carcinoma: an immunohistochemical, ultrastructural, DNA ladder, and TUNEL labeling study.

We performed immunohistochemical, ultrastructural, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and DNA ladder studies of apoptosis in nine cases of Merkel cell carcinoma (MCC). None of the cases showed spontaneous regression as has been reported in several MCCs. Neuron-specific enolase was demonstrated by immunohistochemistry (8/8 MCCs), and staining for cytokeratin 20 was positive (2/8 MCCs). Ultrastructural examination revealed many cytoplasmic dense-cored granules, desmosome-like structures, and intermediate filaments. The granules were seen along the plasma membrane or around perinuclear centrioles. We found various stages of development of apoptotic bodies. Apoptosis resulted in vacuolization and fragmentation of nuclei and phagocytosed bodies in tumor cells. Apoptotic cells were also detected by TUNEL, DNA ladder, and immunostaining using the antibody against Fas (Apo- 1/CD95) antigen. It seems that a high apoptotic rate is a common finding in MCC, although spontaneous regression is an exceedingly rare event. It is thus unlikely that apoptosis alone would explain spontaneous regression.

Cutaneous malakoplakia: a report of two cases with the use of anti-BCG for the detection for micro-organisms.

Malakoplakia is an uncommon granulomatous infectious disease that is found primarily in the genito-urinary tract, but may rarely involve the skin. Histologic findings are marked by the presence of foamy macrophages containing basophilic concentric spherules, the Michaelis-Gutman bodies. Micro-organisms are not readily identifiable. Immunostaining with polyclonal anti-mycobacterium bovis (BCG) has been described as a method of identifying bacterial and fungal organisms in situations where organisms may be sparse. We report 2 cases of cutaneous malakoplakia with demonstration of organisms by immunostaining with anti-BCG antibodies.

Staining of melanocytic neoplasms by melanoma antigen recognized by T cells.

We stained benign melanocytic nevi and malignant melanoma with antibodies to melanoma antigen recognized by T cells (Mart-1) to determine if this was useful in differentiating benign from malignant melanocytic neoplasms. Forty-five primary malignant melanomas and 71 benign melanocytic nevi were stained with antibodies to Mart-1. Two cases of malignant melanoma metastatic to lymph node and three cutaneous metastases of malignant melanoma were also stained. The degree of staining was graded into diffuse positive staining, focal positive staining, and negative staining. Thirty-six of 45 primary malignant melanomas stained diffusely positive with antibodies to Mart-1. This included three of five desmoplastic malignant melanomas that showed positive staining. Four melanomas showed faint or focal positive staining. One of two metastases to lymph node showed strong positive staining and one showed no staining. All three cutaneous metastases showed diffuse positive staining. Sixty-one of 71 melanocytic nevi showed no staining or faint staining with antibodies to Mart-1. Ten of 71 melanocytic nevi showed strong positive staining. The majority of these were congenital nevi. Staining with antibodies to Mart-1 antigen was a useful marker of malignant melanoma. However, staining may also be seen in benign melanocytic neoplasms. The presence or absence of staining for Mart-1 antigen cannot be used to differentiate benign melanocytic nevi from malignant melanocytic tumors.

Cheilitis due to treatment with simvastatin.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are very common and effective treatments for hyperlipidemia. Epidermal cholesterol synthesis has been shown to be essential for maintaining the cutaneous barrier function. We present two patients who experienced cheilitis after beginning treatment of hyperlipidemia with simvastatin (Zocor). The rash resolved after discontinuation of medication and subsequent treatment with topical moisturizers and topical corticosteroids. We suspect that skin barrier dysfunction may occur in the mucosa from inhibitors of HMG-CoA reductase in a manner analogous to the epidermis.

Staining of eccrine and apocrine neoplasms and metastatic adenocarcinoma with IKH-4, a monoclonal antibody specific for the eccrine gland.

The histogenesis of apocrine and eccrine neoplasms has always interested dermatopathologists. In addition, the histologic differential diagnosis of eccrine carcinoma from metastatic adenocarcinoma is of practical importance. We describe a novel monoclonal antibody IKH-4 which stains the eccrine secretory coil, but not the apocrine secretory segment. Positive staining was observed in eccrine hidradenoma, eccrine poroma, eccrine spiradenoma, papillary eccrine adenoma, eccrine hidrocystoma, syringoma, eccrine carcinoma, and in 1 case of syringocystadenoma papilliferum. Negative staining was observed in apocrine adenocarcinoma, hidradenoma papilliferum, erosive adenomatosis of the nipple, and primary and metastatic adenocarcinomas. IKH-4 antibody was useful in differentiating eccrine from apocrine neoplasms and in differentiating eccrine carcinoma from metastatic adenocarcinomas.

Onychomycosis.

Treatment options for onychomycosis are expanding, making accurate and prompt diagnosis of greater importance. Diagnosis of onychomycosis can be made by direct examination of potassium hydroxide-prepared nail clippings, culture, and histologic examination. We compared culture of nail clippings to histologic examination and show that histologic examination is a quick and reliable alternative.

Nodular myxoid change in melanocytic nevi. A report of two cases.

Mucin deposition has been reported in a variety of cutaneous neoplasms, including melanocytic nevi. We describe a series of melanocytic nevi with nodular myxoid change.

Amelanotic lentigo maligna melanoma: a diagnostic conundrum-- presentation of four new cases.

BACKGROUND: The clinical appearance of amelanotic lentigo maligna melanoma (ALMM) is quite confusing and usually is not diagnosed prior to histopathologic examination. METHODS: We have studied four new patients with ALMM whose correct diagnosis was not obtained from any one clinical finding. We arrived at the final diagnosis in an unexpected way, having had biopsied the lesions for diagnoses other than malignant melanoma. RESULTS: ALMM presents as a nonspecific skin lesion with no single indicative characteristic. A search of the literature confirmed our difficulty in making the diagnosis. CONCLUSIONS: To diagnose ALMM, one has to be cognizant of this condition and has to consider a constellation of findings that are unusual with melanocytic lesions. Questionable lesions must be biopsied for definitive histopathologic diagnosis.

Chronic lupoid leishmaniasis. Evaluation by polymerase chain reaction.

BACKGROUND: The cutaneous lesions in chronic lupoid leishmaniasis resemble those of lupus vulgaris, both clinically and histologically. The differential diagnosis is difficult and may depend on the detection of a few Leishmania amastigotes in the histologic sections, the growth of the promastigotes in cultures, or the identification of amastigotes by other techniques. Polymerase chain reaction was used to detect Leishmania amastigote DNA in tissue samples obtained from 65 patients with chronic lupoid leismaniasis, and the results were confirmed by Southern blot analysis. OBSERVATIONS: The histologic findings of a predominantly epithelioid cell granuloma surrounded by lymphocytic infiltrate in chronic lupoid leishmaniasis are very similar to those observed in lupus vulgaris. Extensive histologic examination of the sections in this series revealed occasional macrophages containing a few amastigotes in only 12 cases. Cultures in NNN medium yielded Leishmania promastigotes in 20 cases. Polymerase chain reaction studies using a Leishmania-specific primer identified Leishmania DNA in 30 of 63 cases, and those using a Mycobacterium tuberculosis primer were found to be negative for mycobacteria in 47 cases tested, including 11 cases with a positive tuberculin skin reaction. CONCLUSIONS: The histologic findings in chronic lupoid leishmaniasis resemble those of lupus vulgaris. Polymerase chain reaction studies were useful in identifying amastigotes in 30 (47.6%) of 63 cases. This study confirms the presence of DNA molecules of Leishmania amastigotes in samples of formalin-fixed, paraffin-embedded granulomatous tissue obtained from patients with chronic lupoid leishmaniasis.

Sebaceous epithelioma: a review of twenty-one cases.

BACKGROUND: Sebaceous epithelioma is a benign tumor that must be differentiated from basal cell carcinoma and other appendageal tumors. OBJECTIVE: Histologic and clinical findings were reviewed in 21 patients with sebaceous epitheliomas. METHODS: Twenty-one cases of sebaceous epithelioma collected during a 5-year period were reviewed. RESULTS: The tumors appeared as yellow to flesh-colored papules, most often on the face and neck. They consisted of basaloid cells with foci of sebaceous differentiation. Cystic spaces formed by holocrine degeneration or simulating sebaceous duct lining were common findings. Patients with sebaceous epitheliomas were more likely than the general population to have an internal malignancy. CONCLUSION: Patients with sebaceous epithelioma should be examined for an underlying malignancy.

Spindle cell lipomas. A report of two cases: one with multiple lesions.

BACKGROUND: Spindle cell lipoma is an unusual and histologically distinctive form of lipoma occurring primarily in older individuals. OBJECTIVE: The dermatologic surgeon and pathologist should be familiar with this variant of lipoma. METHODS: We are reporting two cases of spindle cell lipomas with the results of histologic examination with special stains and immunohistochemical studies. RESULTS: The neoplasms consisted of lobulated masses of mature adipose tissue with areas of spindle cell proliferation. There was strong positive staining with antibodies for vimentin within the areas of spindle cells proliferation. One of the cases is unusual in that multiple lesions are occurring in a young patient. CONCLUSION: Recognition of the histologic pattern is important in distinguishing the benign spindle cell lipoma from the malignant liposarcoma.

Axillary granular parakeratosis.

We report two cases of axillary granular parakeratosis, which is a unique eruption involving the axilla that has distinctive histopathologic features. Both of our patients had slightly pruritic, hyperpigmented patches in the axilla. The biopsy specimens revealed severe compact parakeratosis with maintenance of the stratum granulosum and retention of keratohyalin granules throughout the stratum corneum, which was markedly thickened and measured between 90 to 185 microns. The exact etiology is not known, but this conditions is believed to represent a contact reaction to an antiperspirant or deodorant.