Applications of pathology-assisted image analysis of immunohistochemistry-based biomarkers in oncology.

Immunohistochemistry-based biomarkers are commonly used to understand target inhibition in key cancer pathways in preclinical models and clinical studies. Automated slide-scanning and advanced high-throughput image analysis software technologies have evolved into a routine methodology for quantitative analysis of immunohistochemistry-based biomarkers. Alongside the traditional pathology H-score based on physical slides, the pathology world is welcoming digital pathology and advanced quantitative image analysis, which have enabled tissue- and cellular-level analysis. An automated workflow was implemented that includes automated staining, slide-scanning, and image analysis methodologies to explore biomarkers involved in 2 cancer targets: Aurora A and NEDD8-activating enzyme (NAE). The 2 workflows highlight the evolution of our immunohistochemistry laboratory and the different needs and requirements of each biological assay. Skin biopsies obtained from MLN8237 (Aurora A inhibitor) phase 1 clinical trials were evaluated for mitotic and apoptotic index, while mitotic index and defects in chromosome alignment and spindles were assessed in tumor biopsies to demonstrate Aurora A inhibition. Additionally, in both preclinical xenograft models and an acute myeloid leukemia phase 1 trial of the NAE inhibitor MLN4924, development of a novel image algorithm enabled measurement of downstream pathway modulation upon NAE inhibition. In the highlighted studies, developing a biomarker strategy based on automated image analysis solutions enabled project teams to confirm target and pathway inhibition and understand downstream outcomes of target inhibition with increased throughput and quantitative accuracy. These case studies demonstrate a strategy that combines a pathologist's expertise with automated image analysis to support oncology drug discovery and development programs.

Photoaging: the role of oxidative stress.

Our skin suffers from daily exposure to oxidative stress, primarily from exposure to the sun's damaging ultraviolet (UV) rays. This results in the appearance of premature aging. The mechanisms of this photodamage to all layers of the skin by the various wavelengths (UVB and UVA) will be clarified. Recent research further demonstrates a synergistic enhancement of oxidative damage when the skin is exposed to UVA in combination with environmental urban pollutants (including cigarette smoke). The fact that photoaging is largely caused by oxidative damage is confirmed by the demonstration that treatment with topical antioxidants can prevent and even partially reverse UV-induced photodamage. Research substantiating the efficacy of vitamins C and E and of selenium will be reviewed.

Synergistic damage by UVA radiation and pollutants.

Not only is skin cancer by far the most common human cancer but also the incidence of skin cancer has been increasing at an alarming rate in recent decades. Fortunately, most people now realize that sun exposure causes unattractive photoaging and skin cancer, so they do apply sunscreens conscientiously. However, until recently, most sunscreens did not adequately protect against ultraviolet A (UVA) radiation. Although UVA is indeed less erythrogenic and less carcinogenic than UVB, UVA directly causes photoaging and enhances UVB-induced skin cancer. Furthermore, recent research demonstrates that UVA combined with environmental pollutants (including cigarette smoke) significantly increases the risk of skin cancer. Similarly, previous research demonstrated another synergy between environmental pollutants and UV: When ozone exposure precedes UV exposure, there is enhancement of UV-induced depletion of protective vitamin E from the skin's stratum corneum. This article reviews experimental evidence that environmental pollutants (such as benzo[a]pyrene (BaP), a commonly used index of environmental pollution) are photosensitizers that generate reactive oxygen species (ROS) when exposed to UVA radiation. This in turn causes oxidative and genetic damage, leading to unattractive photodamage and carcinogenesis.

Photodamage of the skin: protection and reversal with topical antioxidants.

Controversy exists as to whether topical antioxidants can be effective in protecting against and reversing photodamage to the skin. Topical vitamins C and E, as well as topical selenium, protect skin against sunburn, suntan and skin cancer and also reverse the mottled pigmentation and wrinkles of photoageing. However, only certain forms of these labile antioxidants are stable and active after percutaneous absorption. For effective topical application, vitamin C must be non-esterified, acidic and optimally at 20% concentration; vitamin E must be the non-esterified isomer d-alpha-tocopherol at 2-5% concentration. Selenium is only percutaneously absorbed and active when applied topically as l-selenomethionine, optimally at 0.02-0.05%. There are two great advantages in applying an active formulation of topical antioxidants to the skin. First, the skin attains far higher levels of each antioxidant than can be achieved by only taking these vitamins orally. The level of vitamin C attained in the skin by topical application is 20-40 times that achievable with oral vitamin C. With topical application, the concentration of vitamin E in the skin increases by a factor of 10.6 and selenium by a factor of 1.7. Second, topical application arms the skin with a reservoir of antioxidants that cannot be washed or rubbed off, a protection which stays in the skin for several days after application.

Effects of topical and oral vitamin E on pigmentation and skin cancer induced by ultraviolet irradiation in Skh:2 hairless mice.

This study investigates whether supplementation with topical RRR-alpha-tocopherol (Eol), topical RRR-alpha-tocopheryl succinate, and oral RRR-alpha-tocopheryl acetate can reduce the incidence of acute and chronic damage to the skin (i.e., sunburn and pigmentation and skin cancer, respectively) induced by ultraviolet (UV) irradiation to mice. Groups of twenty Skh:2 female hairless pigmented mice were treated with 1) lotion vehicle, 2) 5% Eol lotion, 3) 5% topical RRR-alpha-tocopheryl succinate lotion, or 4) lotion vehicle and oral RRR-alpha-tocopheryl acetate. Within each group, 15 mice were exposed to 0.24 J/cm2 of UV-B radiation three times per week. The animals' weights and food intakes were monitored, and the vitamin E concentrations of skin, liver, and adipose tissue were measured to determine whether the topical Eol resulted in significant tissue levels. Skin pigmentation was scored, and the total number of clinically detectable skin tumors per animal was counted weekly. Results showed that the skin concentrations of Eol, as well as levels in the adipose tissue, were increased after topical application. Mice treated with each form of vitamin E showed no signs of toxicity and had significantly less acute and chronic skin damage induced by UV irradiation, as indicated by reduced inflammation and pigmentation and by later onset and lesser incidence of skin cancer.

Human subcutaneous adipose tissue shows site-specific differences in fatty acid composition.

Adipose tissue was obtained from six women undergoing liposuction twice at 6-mo intervals. Samples obtained bilaterally from abdomen, inner thigh, and outer thigh had fatty acids quantified by gas chromatography. There were no important differences between sides or over time. The saturates 14:0, 16:0, 18:0, and 20:0 were higher in abdominal adipose than in outer thigh (P < 0.002 for all); 16:1 and 18:1 omega 9 were lower in abdomen vs outer thigh (P < 0.01), whereas 18:1 omega 7 and 20:1 omega 9 were unchanged. Polyunsaturates 18:2 omega 6, 20:3 omega 6, and 20:4 omega 6 were higher in outer thigh than in abdomen (P < 0.06), and inner thigh values were intermediate. These changes in fatty acid composition resulted in lower mean triglyceride melting points from abdomen to inner thigh to outer thigh, and suggest that temperature may influence the selection process determining the variation in adipose fatty acid composition with anatomical location. Because the site-specific differences included essential fatty acids, selective uptake as well as potential differences in in situ fatty acid modification are indicated.

The effect of topical L-selenomethionine on minimal erythema dose of ultraviolet irradiation in humans.

The purpose of this study was to determine whether topical L-selenomethionine reduces the degree of acute damage to the skin (i.e., sunburn) induced by ultraviolet (UV) irradiation in humans. Eight women volunteers were treated for 2 weeks first with lotion vehicle then with topical L-selenomethionine for two weeks at each of three concentrations (0.002%, 0.02%, and 0.05%). At the end of each 2-week treatment period, the minimal erythema dose (MED) of UV irradiation was measured using a Multiport Solar Ultraviolet Simulator. It was found that topical L-selenomethionine was effective in protecting against acute UV damage to the skin. A response curve demonstrated that increasing concentrations of topical L-selenomethionine gave increasing MED, with a plateau of maximal protection attained at a concentration between 0.02% and 0.05%. Although previous studies in rats and mice demonstrated percutaneous absorption of selenium after application of L-selenomethionine, in the concentrations used here, the topical L-selenomethionine did not result in increased levels of selenium in the blood plasma of the patients tested.

The effects of topical and oral L-selenomethionine on pigmentation and skin cancer induced by ultraviolet irradiation.

This study was conducted to determine whether oral and/or topical selenium (Se) supplementation can reduce the incidence of acute and/or chronic damage to the skin (i.e., sunburn and pigmentation and/or skin cancer, respectively) induced by ultraviolet (UV) irradiation in mice. Groups of 38 BALB:c female mice or 16 Skh:2 hairless pigmented mice were treated with 1) lotion vehicle, 2) 0.02% L-selenomethionine (SeMet) lotion, or 3) vehicle and 1.5 ppm SeMet in the drinking water. Within each group, 30 BALB:c mice or 12 Skh:2 mice were given UV irradiation (Westinghouse FS 40 bulbs) three times per week in doses of 0.575 and 0.24 J/cm2, respectively. The animals' weights and food intakes and the Se concentrations of skin and liver were measured. Skin biopsies were taken from the backs and abdomens of all animals to evaluate the relative amounts of Se and the damage by UV irradiation. Skin pigmentation was scored, and the total number of clinically detectable skin tumors per animal was counted weekly. Results showed that the skin Se concentrations in areas of application of the lotion containing SeMet were greater than those of animals given comparable oral doses, while the Se concentrations of untreated skin and liver were similar to those of animals receiving oral Se. Mice treated with Se showed no signs of toxicity and had significantly less skin damage by UV irradiation, as indicated by reduced inflammation and pigmentation and by later onset and lesser incidence of skin cancer.

Facial wrinkles. Prevention and nonsurgical correction.

There is no way to age without getting wrinkles. However, patients can be taught how to retard the onset of wrinkles by avoiding unprotected sun exposure, unnecessary facial movements, and certain sleeping positions. Relatively simple non-surgical techniques are available for correction of wrinkles. Small wrinkles can be treated with exfoliation, topical tretinoin (Retin-A), or topical alpha hydroxy acids. Large wrinkles can be improved with implantation of bovine collagen, silicone, or fat extracted from the abdomen or thigh of the patient. Each method is effective when used for the correct indications.

Hair loss. What causes it and what can be done about it.

Although both men and women throughout history have seen hair as an important aspect of appearance, it is especially important today, in light of the great emphasis on youthfulness. A new interest in preventing baldness has been stimulated recently by the publicity given to certain products now under investigation that have shown an ability to retard or reverse male pattern baldness in certain individuals. Hair loss has many possible causes, such as systemic diseases, infections, toxic agents, and hormone imbalances. Treatment of the underlying disorder alleviates the shedding of hair. Balding may also be a normal physiologic occurrence in women taking oral contraceptives or after parturition and in men with male pattern baldness. The latter can be treated topically with progesterone or minoxidil. Minoxidil has been studied extensively and has been shown to improve balding at the vertex of the scalp, particularly in young men who have only begun to lose hair. Cases of more extensive male pattern baldness and baldness secondary to scarring can be treated effectively with surgical procedures.

A histological, immunological, and electron microscopic study of bovine collagen implants in the human.

The histological fate of an injectable form of type I bovine dermal collagen (Zyderm collagen implant) in the human dermis and subcutaneous tissue has been studied. Sequential biopsies were analyzed by hematoxylin-eosin and Weigert's stains, by immunofluorescence (using a highly specific rabbit anti-Zyderm collagen antibody and a specific anti-human type III collagen antibody), and by electron miscroscopy. The results of this study suggest that the bovine implant material stimulates a host response resulting in implant degradation and replacement by newly generated host collagen.

Bovine collagen implant: histologic chronology in pig dermis.

The histologic fate of an injectable form of type I bovine dermal collagen (Zyderm Collagen Implant, ZCI) in the pig dermis and subcutaneous tissue has been studied. Biopsies were taken sequentially and analyzed by hematoxylin and eosin (H&E) and Weigert's stain, by birefringence, by direct immunofluorescence (using a highly specific rabbit anti-Zyderm antibody), and by electron microscopy. The implant material was found to stimulate a host response resulting in implant degradation and replacement by newly generated host collagen.

Determination of microgram quantities of silver in aluminium-, iron-, and nickel-base alloys.

Trace quantities of silver in commercial nickel and its alloys are quantitatively extracted from 10% hydrochloric acid medium containing 2% ascorbic acid and 9% potassium iodide by a 5% solution of tri-n-octylphosphine oxide in 4-methylpentan-2-one (MIBK). The MIBK-extract containing the silver is then nebulized directly into an atomic-absorption flame. The sensitivity for the determination of silver in MIBK is about 6 times that for aqueous media. The proposed technique is accurate, rapid, and has a standard deviation of +/- 0.025 ppm at the 1 ppm level. The limit of detection is 0.2 ppm of silver. The method is also applicable to a number of elements in aluminium- and iron-base alloys.

A chemical concentration x-ray determination of selenium in copper-, nickel-and iron-base alloys.

A method is described for the determination of selenium in various metals and alloys at concentrations of 3-100ppm. A chemical separation is made by filtration after reduction of the selenious acid to elemental selenium with hydroxylamine hydrochloride. The pure selenium product is readily analysed by X-ray fluorescence; the method is free from errors due to matrix effects. The relative standard deviation is 6 % for a copper-nickel alloy at the 60 ppm level.

Determination of boron in nickel-base alloys by the dianthrimide method.

The dianthrimide method for the determination of boron in iron and low-alloy steels may be applied to nickel-base materials. The sample is dissolved, without any loss of boron, by hydrochloric and sulphuric acids and the resulting boric acid determined spectrophotometrically with dianthrimide. Background corrections are necessary to compensate for the absorbance from ions such as nickel and iron.