Application of next-generation sequencing to improve cancer management: A review of the clinical effectiveness and cost-effectiveness.

Uptake of next-generation sequencing (NGS) has increased dramatically due to significant cost reductions and broader community acceptance of NGS. To systematically review the evidence on both the clinical effectiveness and the cost-effectiveness of applying NGS to cancer care. A systematic search for full-length original research articles on the clinical effectiveness and cost-effectiveness of NGS in MEDLINE and EMBASE. Articles that focussed on cancer care and involved the application of NGS were included for the review of clinical effectiveness. For the cost-effectiveness review, we only included the articles with economic evaluations of NGS in cancer care. We report the rate of successfully detecting mutations from the clinical studies. The incremental cost-effectiveness ratio and sensitivity analysis outcomes are reported for the cost-effectiveness articles. Fifty-six articles reported that sequencing patient samples using targeted gene panels, and 83% of the successfully sequenced patients harboured at least 1 mutation. Only 6 studies reported on the cost-effectiveness of the application of NGS in cancer care. NGS is an effective tool for identifying mutation in cancer patients. However, more rigorous cost-effectiveness studies of NGS applied to cancer management are needed to determine whether NGS can improve patient outcomes cost-effectively.

High sensitivity SnO2 single-nanorod sensors for the detection of H2 gas at low temperature.

Uniform SnO(2) nanorods were grown by inductively coupled plasma-enhanced chemical vapor deposition without catalysts and additional heating. The SnO(2) nanorods were aligned on a pair of Au/Ti electrodes by the dielectrophoresis method. SnO(2) single-nanorod gas sensors were fabricated by connecting individual SnO(2) nanorods to a pair of Au/Ti electrodes with Pt stripes deposited by a focused ion beam. The sensing properties of the SnO(2) single-nanorod sensor were studied. The SnO(2) single-nanorod sensor could detect 100 ppm H(2) at room temperature with repeated response and showed a large change of resistance, fast response time and good reversibility at an elevated operating temperature of 200 degrees C. The optimal sensing performance of the sensor is achieved at the operating temperature of around 250 degrees C.

Regulation and activation of ezrin protein in endometriosis.

BACKGROUND: Ezrin protein and its activated form phospho-ezrin play a role in cell morphology, motility and adhesiveness. In this study, we hypothesized that these proteins play a role in the pathogenesis of endometriosis by promoting adhesion and invasion of endometrial stromal cells (ESCs) in ectopic sites. METHODS: We compared the expression of ezrin and phospho-ezrin in normal endometrium from women without endometriosis with their expression in eutopic and ectopic endometrial tissues from women with endometriosis, using immunohistochemistry and western blot analysis. Paired eutopic and ectopic endometrial tissue samples from women with endometriosis (n = 13) and normal endometrium from women without endometriosis (n = 12) were collected. Invasive potential of ESCs from each of these samples was compared using Matrigel membrane invasion assay. RESULTS: Eutopic and ectopic endometrial tissues from women with endometriosis have higher ezrin and phospho-ezrin levels as confirmed by immunohistochemistry and western blot analysis (P < 0.05). The Matrigel membrane invasion assay revealed that ectopic ESCs have more invasive characteristics, more protrusions and higher ezrin staining than normal ESCs (P < 0.05). CONCLUSIONS: Ezrin can be a potential marker for endometrial cell invasion and may play a role in the pathogenesis of endometriosis.

Aqueous humor endothelin-1 and total retinal blood flow in patients with non-proliferative diabetic retinopathy.

PurposeThe purpose of this study was to determine the association between aqueous ET-1 levels and total retinal blood flow (TRBF) in patients with non-insulin-dependent type 2 diabetes mellitus (T2DM) and early non-proliferative diabetic retinopathy (NPDR).Patients and methodsA total of 15 age-matched controls and 15 T2DM patients with NPDR were recruited into the study. Aqueous humor (~80-120 µl) was collected before cataract surgery to measure the levels of ET-1 using suspension multiplex array technology. Four weeks post surgery, six images were acquired to assess TRBF using the prototype RTVue Doppler FD-OCT (Optovue, Inc., Fremont, CA, USA) with a double circular scan protocol. At the same visit, forearm blood was collected to determine plasma glycosylated hemoglobin (A1c) levels.ResultsAqueous ET-1 was significantly elevated in the NPDR group compared with the control group (3.5±1.8 vs 2.2±0.8, P=0.02). TRBF was found to be significantly reduced in the NPDR group compared with the control group (34.5±9.1 vs 44.1±4.6 µl/min, P=0.002). TRBF and aqueous ET-1 were not correlated within the NPDR group (r=-0.24, P=0.22). In a multivariate analysis, high A1c was associated with reduced TRBF and aqueous ET-1 levels across control and NPDR groups (P<0.01).ConclusionAqueous ET-1 levels were increased while TRBF was reduced in patients with NPDR compared with the control group. Although not directly associated, the vasoconstrictory effects of ET-1 are consistent with a reduced TRBF observed in early DR. ET-1 dysregulation may contribute to a reduction in retinal blood flow during early DR.

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms.

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

Effect of skin temperature on selective vascular injury caused by pulsed laser irradiation.

The effect of skin temperature on vascular-specific injury caused by pulsed laser irradiation was examined. Ten healthy human volunteers were exposed to 1.5 microsecond pulses from a dye laser tuned to 577 nm. Compared to normothermic conditions (33 degrees C skin temperature) significantly more laser energy (p less than 0.01) was required to produce grossly visible purpura when the skin was cooled to 20 degrees C or heated to 40 degrees C. Histologically, laser-induced damage was confined to blood vessels at all three skin temperatures studied. At purpura threshold dose, there was intravascular agglutination without extravasation of red blood cells at 20 degrees C whereas at 33 degrees and 40 degrees C there was extravasation of red blood cells.

Ultrastructural changes in human skin after exposure to a pulsed laser.

Selective vascular injury following irradiation using a pulsed laser source at 577 nm was examined using ultrastructural methods in the skin of 3 fair-skinned healthy human volunteers. This vascular-specific damage was confined to the papillary dermis. Red blood cells were altered in several ways. As well as an increase in the electron density, configurational distortion modified the normal biconcave forms to ameboid structures. The most interesting finding was the appearance within these altered cells of well-defined circular/oval electron-lucent areas of 800 A diameter, possibly representing a heat-fixed record of steam formation within the red blood cell. In addition, considerable degenerative changes were evident in endothelial cells and pericytes, while mast cells, neutrophils, histiocytes, and fibroblasts as well as collagen bundles immediately surrounding most laser-damaged blood vessels appeared normal.

Action spectrum of vascular specific injury using pulsed irradiation.

It has been clearly demonstrated that cutaneous blood vessels will be selectively damaged by a laser whose wavelength matches one of the three absorption spectral peaks of the chromophore, oxyhemoglobin, for example, 577 nm. A restriction in the application of this wavelength for the treatment of benign cutaneous vascular tumors, such as portwine stains, has been the penetration depth of 577 nm irradiation of approximately 0.5 mm from the dermal epidermal junction (DEJ). This study was undertaken to establish whether it was possible to increase the penetration depth from 0.5 mm by changing the wavelength to beyond 577 nm in albino pig skin. Results from this study confirm that penetration depth increases from 0.5 to 1.2 mm by changing the wavelength from 577 to 585 nm at 4 J/cm2, while maintaining the same degree of vascular selectivity as that previously described after 577 nm irradiation. This occurred in spite of a mismatch in the wavelength between 585 nm and the oxyhemoglobin absorption peak of 577 nm. Unlike 585 nm irradiation and in contrast with theoretical predictions, 590 nm laser light did not penetrate as deeply as 585 nm. Not only was there a reduction in the penetration depth of the laser beam from 1.2 mm at 585 nm to 0.8 mm at 590 nm, at 4 J/cm2, but there was also a decrease in vascular selectivity in albino pig skin exposed to 590 nm irradiation.

193 nm excimer laser selective ablation of in vivo guinea pig epidermis.

We have previously shown that 193 nm excimer laser irradiation cleanly and effectively ablates avascular tissue with minimal thermal damage to surrounding adjacent structures. In this study, the 193 nm excimer laser is used to remove guinea pig epidermis in vivo. The epidermis can be totally ablated with thermal damage extending only superficially into the dermis. Reepitheliazation of the ablated area takes place in 1 week or less. This technique may be applicable to the removal of benign epidermal lesions.

Pulsed dye laser (577 nm) treatment of portwine stains: ultrastructural evidence of neovascularization and mast cell degranulation in healed lesions.

Portwine stains were examined before, immediately after, and 1 yr after successful clearance by a pulsed dye laser (577 nm) using ultrastructural techniques. Dilated vascular channels and mast cell hypoplasia characterized lesional skin before treatment. Immediately after treatment, widespread selective vessel necrosis, similar to changes previously described, was observed. One year after laser irradiation, the abnormally ectatic portwine stain vessels had been replaced by small venules and arterioles, similar in number and diameter to blood vessels in normal skin; the only difference noted was that these new vessels were surrounded by easily identifiable mast cells. Many of these mast cells exhibited evidence of activation and degranulation. We conclude that mast cells may play an important role in the neovascularization of portwine stains treated by 577-nm dye laser irradiation.

Spotsize effects on guinea pig skin following pulsed irradiation.

Laser irradiation parameters such as wavelength, irradiance (W/cm2), and pulse duration have been clearly shown to influence the extent to which tissue is damaged. The careful choice of these parameters can result in confining laser injury to specific targets in tissue. Spotsize, a parameter not commonly appreciated in the application of lasers to medicine and surgery, has been shown, in this study, to contribute to the ultimate outcome of laser effects in tissue. A series of histological events occurring in the skin are demonstrated to be directly related to the effects of spotsize on tissue at a fixed exposure time and wavelength. Many of these changes could contribute to unwanted adverse effects, such as scarring, which occur following certain laser therapies.

Effect of cutaneous hypoxia upon erythema and pigment responses to UVA, UVB, and PUVA (8-MOP + UVA) in human skin.

The effect of oxygen deprivation upon UVA-, UVB-, and PUVA-induced pigment and erythema responses in normal human skin was examined. Before exposure, varying degrees of hypoxia in the skin of the forearm were achieved by inflating a sphygmomanometer cuff applied to the upper arm. After the transcutaneously measured pO2 had stabilized, sites on the inner forearm were exposed to UVA, UVB, or 8-MOP + UVA radiation, to determine dose thresholds for the induction of erythema and pigmentation at different cuff pressures. Inflation of the cuff to greater than systolic pressure completely inhibited immediate and delayed pigment responses (IPD, DT) to UVA doses greater than 10 times the normal pigmentation threshold dose. UVA-induced delayed erythema responses were partially inhibited by cuff inflation: 2.7 times the minimal erythema dose of UVA was necessary to cause an erythema response when exposure occurred during vascular occlusion. In contrast, erythema and pigment responses to UVB and PUVA were unaltered by cuff pressures exceeding systolic pressure during exposure. Inhibition of UVA-induced erythema and pigment responses by vascular occlusion were reversed by the transcutaneous diffusion of 100% O2. These findings indicate that the cutaneous responses to UVA and UVB occur by separate pathways differing with respect to O2 dependence. Our findings agree with those of other studies which indicate that PUVA-induced phototoxicity and melanogenesis are not O2-dependent.

Effect of wavelength on cutaneous pigment using pulsed irradiation.

Several reports have been published over the last two decades describing the successful removal of benign cutaneous pigmented lesions such as lentigines, café au lait macules' nevi, nevus of Ota, and lentigo maligna by a variety of lasers such as the excimer (351 nm), argon (488,514 nm), ruby (694 nm), Nd:YAG (1060 nm), and CO2 (10,600 nm). Laser treatment has been applied to lesions with a range of pigment depths from superficial lentigines in the epidermis to the nevus of Ota in the reticular dermis. Widely divergent laser parameters of wavelength, pulse duration, energy density, and spotsizes have been used, but the laser parameters used to treat this range of lesions have been arbitrary, with little effort focused on defining optimal laser parameters for removal of each type. In this study, miniature black pig skin was exposed to five wavelengths (504, 590, 694, 720, and 750 nm) covering the absorption spectrum of melanin. At each wavelength, a range of energy densities was examined. Skin biopsies taken from laser-exposed sites were examined histologically in an attempt to establish whether optimal laser parameters exist for destroying pigment cells in skin. Of the five wavelengths examined, 504 nm produced the most pigment specific injury; this specificity being maintained even at the highest energy density of 7.0 J/cm2. Thus, for the destruction of melanin-containing cells in the epidermal compartment, 504 nm wavelength appears optimal.

Ultrastructure: effects of melanin pigment on target specificity using a pulsed dye laser (577 nm).

It has been shown recently that brief pulses of 577 nm radiation from the tunable dye laser are absorbed selectively by oxyhemoglobin. This absorption is associated with highly specific damage to superficial vascular plexus blood vessels in those with lightly pigmented (type I-II) skin. To determine whether pigmentary differences in the overlying epidermis influence this target specificity, we exposed both type I (fair) and type V (dark) normal human skin to varying radiant exposure doses over 1.5-microsecond pulse durations from the tunable dye laser at a wavelength of 577 nm. Using ultrastructural techniques, we found in type I skin that even clinical subthreshold laser exposures caused reproducible alterations of erythrocytes and adjacent dermal vascular endothelium without comparable damage to the overlying epidermis. In contrast, degenerated epidermal basal cells represented the predominant form of cellular damage after laser exposure of type V skin at comparable doses. We conclude that epidermal melanin and vascular hemoglobin are competing sites for 577 nm laser absorption and damage, and that the target specificity of the 577 nm tunable dye laser is therefore influenced by variations in epidermal pigmentation. This finding is relevant to the clinical application of the tunable dye laser in the ablative treatment of vascular lesions. We also found on ultrastructure that the presence of electron-lucent circular structures of approximately 800 A in diameter were observed only at and above clinical threshold doses in those with type I skin and at the highest dose of 2.75 J/cm2 in type V skin. It has been proposed that these structures might be heat-fixed molds of water vapor. Both this and ultrastructural changes of epidermal basal cells demonstrate mechanisms responsible for alteration of tissue after exposure to 577 nm, which are discussed.

Ultraviolet excimer laser ablation: the effect of wavelength and repetition rate on in vivo guinea pig skin.

Multiple dermatologic conditions that are currently treated with traditional cold-knife surgery are amenable to laser therapy. The ideal surgical treatment would be precise and total removal of abnormal tissue with maximal sparing of remaining structures. The ultraviolet (UV) excimer laser is capable of such precise tissue removal due to the penetration depth of 193 nm and 248 nm irradiation of 1 micron per pulse. This type of ablative tissue removal requires a high repetition rate for efficient lesional destruction. Excimer laser radiation at 193 nm is capable of high repetition rates, which are necessary while 248 nm radiation causes increasing nonspecific thermal injury as the laser repetition rate is increased.

Ultrastructural changes in red blood cells following pulsed irradiation in vitro.

The careful choice of a combination of laser parameters such as wavelength, pulse duration, and dose has provided a means for confining laser energy to specific targets within tissue such as oxyhemoglobin within the cutaneous microvasculature. In the process of achieving such vascular selectivity, certain ultrastructural changes in red blood cell (RBC) cytoplasm have been observed, such as the generation of intracytoplasmic electron-lucent spherical structures. These structures, ranging in size from 80 to 1000A, were seen in RBCs exposed to laser doses at and above threshold, and appeared to represent a morphologically novel form of highly-specific tissue injury. This in vitro study using RBC in phosphate buffered saline (PBS) was undertaken to better understand the mechanism(s) that could have been responsible for these unique morphologic changes. We conclude that the intracytoplasmic electron-lucent spherical structures seen within RBCs were heat-fixed molds formed around vaporized water bubbles and were not produced by the release of oxygen from the oxyhemoglobin moiety during 577-nm laser irradiation.

Effect of dye laser pulse duration on selective cutaneous vascular injury.

The pulsed dye laser at 577 nm, a wavelength well absorbed by oxyhemoglobin, causes highly selective thermal injury to cutaneous blood vessels. Confinement of thermal damage to microvessels is, in theory, related to the laser exposure time (pulsewidth) on selective vascular injury. This study investigates the effect of 577 nm dye laser pulsewidth on selective vascular injury. Nine Caucasian, normal volunteers received 577 nm dye laser exposures at pulsewidths of 1.5-350 microseconds to their skin. Clinical purpura threshold exposure doses were determined in each volunteer, and biopsies of threshold and suprathreshold doses were examined in each volunteer. The laser exposure dose required to produce purpura increased as pulsewidth increased in all 9 subjects (p less than 0.001). This finding corresponds to laser pulsewidths equal to or exceeding the thermal relaxation times for dermal blood vessels. Histologically, vessel damage was selectively, but qualitatively, different for short vs long pulsewidths. Pulsewidths shorter than 20 microseconds caused vessel wall fragmentation and hemorrhage, whereas longer pulsewidths caused no significant hemorrhage. The purpura noted clinically appears to be due to a coagulum of intralumenal denatured erythrocytes. At 24 h, there was marked vessel wall necrosis at all pulsewidths. The short pulsewidths may cause erythrocyte vaporization, rapid thermal expansion, and mechanical vessel rupture with hemorrhage. Long pulsewidths appear to cause thermal denaturation with less mechanical vessel damage. The selective, nonhemorrhagic, vascular necrosis caused by the long-pulsewidth dye laser may lead to a more desirable clinical outcome in the therapy of blood vessel disease processes.

Factitious dermatosis masquerading as recurrent herpes zoster.

A 35-year-old nurse's aide presented with monthly episodes, during her menses, of self-induced cutaneous lesions intended to simulate recurrent herpes zoster. Features of the clinical presentation that prompted the correct diagnosis are discussed.

Congo red binding of elastin in aortic smooth muscle cell cultures.

These studies demonstrate that the strong binding capacity of elastin for Congo red can be used to advantage in aortic smooth muscle cell cultures. A fibrous elastin network fluoresces when Congo red is added. Congo red does not alter accumulation of elastin or of total protein, even when the cells are grown in the presence of the dye for long periods of time, indicating that it is not toxic. Porcine pancreatic elastase was used to solubilize elastin in these cultures, to determine the molar ratio of Congo red to elastin, thus making it possible to estimate the amount of elastin solubilized when the cultures are injured. Congo red binding to elastin will be useful in studying elastin accumulation and/or degradation in vitro and in vivo.

Laser therapy for selected cutaneous vascular lesions in the pediatric population: a review.

Three cutaneous vascular lesions of childhood (the spider angioma [nevus araneus], the strawberry hemangioma, and the port wine stain) are reviewed, with particular emphasis on the present and future role of laser therapy in their management.