Use of PRAME immunostaining to distinguish early melanoma in situ from benign pigmented conditions.

BACKGROUND: PRAME (PReferentially expressed Antigen in MElanoma) is an antigen that shows marked overexpression in melanoma compared to normal skin melanocytes. PRAME immunohistochemistry has proven effective in distinguishing melanocytic nevi from melanoma, but it is unclear if it may be used to distinguish melanoma in situ from other benign pigmented lesions. In particular, differentiating from melanocytic hyperplasia in sun-damaged skin is sometimes clinically and histopathologically challenging. We hypothesized that PRAME staining of solar lentigo, sun-damaged skin, and melanoma in situ would aid in setting a threshold of positivity that could be useful in evaluating such conditions. METHODS: We collected and stained typical examples of solar lentigo, melanoma in situ, and non-lesional sun-damaged skin by PRAME immunohistochemistry to assess a potential cutoff of PRAME positivity. RESULTS: Solar lentigo and non-lesional sun-damaged skin had 10 or fewer PRAME-positive cells per millimeter (mean 1.2), on the other hand melanoma in situ had at least 16 (mean 75.1). CONCLUSIONS: PRAME immunostaining appears sensitive and specific in the current series. This could be clinically useful for distinguishing melanoma in situ from benign melanocytic hyperplasia in sun-damaged skin. However, further studies are required to determine if 10 cells per millimeter is an acceptable threshold of positivity.

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#InSituPathologists: how the #USCAP2015 meeting went viral on Twitter and founded the social media movement for the United States and Canadian Academy of Pathology.

Professional medical conferences over the past five years have seen an enormous increase in the use of Twitter in real-time, also known as "live-tweeting". At the United States and Canadian Academy of Pathology (USCAP) 2015 annual meeting, 24 attendees (the authors) volunteered to participate in a live-tweet group, the #InSituPathologists. This group, along with other attendees, kept the world updated via Twitter about the happenings at the annual meeting. There were 6,524 #USCAP2015 tweets made by 662 individual Twitter users; these generated 5,869,323 unique impressions (potential tweet-views) over a 13-day time span encompassing the dates of the annual meeting. Herein we document the successful implementation of the first official USCAP annual meeting live-tweet group, including the pros/cons of live-tweeting and other experiences of the original #InSituPathologists group members. No prior peer-reviewed publications to our knowledge have described in depth the use of an organized group to "live-tweet" a pathology meeting. We believe our group to be the first of its kind in the field of pathology.

CD30 Expression Is Rare in Myeloid Leukemia Cutis: A Study of 55 Cases and Implications for Routine Diagnostic Algorithms.

Expression of CD30 in blastoid cutaneous infiltrates typically signifies a CD30 lymphoproliferative disorder, often requiring minimal immunohistochemical workup, if clinically consonant. However, myeloid and other hematologic malignancies often express CD30. We retrospectively examined the prevalence of CD30 expression in 41 patients (median age 59) and 55 biopsies with the diagnosis of leukemia cutis (LC) to determine whether an extensive immunohistochemical workup is warranted in all large, round cell CD30 cutaneous infiltrates. Each patient had refractory or recurrent disease, the histologic presence of a large mononuclear cell infiltrate, and varied cytogenetics. CD30 mononuclear cells within the infiltrate ranged from rare to many in 22 biopsies (22/55). In 18 biopsies, CD30 cells were interpreted as lymphocytic based on morphology, strong cytoplasmic and Golgi staining for CD30, and negative CD34 and CD117 staining. One case showing 3+ staining of lymphocytes was identified as a posttransplant lymphoproliferative disorder. The second 3+ case was favored to represent a subset of CD30-positive acute myeloid leukemia. Three other cases with 1+ membranous and cytoplasmic staining were interpreted as myeloid leukemia. In conclusion, CD30 positivity in myeloid leukemia in the skin is rare and does not often exhibit the strong membranous (2+ or 3+) and/or Golgi staining seen in reactive lymphocytes. Acute myeloid leukemia or myeloid LC may occasionally show 1+ (and rarely 2-3+) cytoplasmic/membranous or nonspecific blush nuclear CD30 labeling. Strong diffuse staining for CD30 should prompt consideration of a reactive lymphoid/lymphoproliferative process, and, when the clinical likelihood of CD30 LC is low, may obviate the need for further immunohistochemistry.

A Comparative Study to Evaluate Epidermal Barrier Integrity of Psoriasis Patients Treated With Calcipotriene/Betamethasone Topical Suspension Versus Betamethasone Dipropionate 0.05% Lotion.

BACKGROUND: Topical corticosteroids are known to impair the epidermal barrier, even after short-term use, whereas topical vitamin D analogues can have a reparative effect. Combination products using corticosteroids and vitamin D analogues have gained popularity in recent years and may provide a means to minimize skin atrophy in patients treated with topical corticosteroids. OBJECTIVE: To compare epidermal barrier function and cutaneous atrophy after 4 weeks of calcipotriene 0.005% and betamethasone dipropionate 0.064% topical suspension (Taclonex® TS) versus betamethasone dipropionate 0.05% lotion (Diprosone®). METHODS: Ten subjects with moderate plaque psoriasis were enrolled. Patients were randomized to apply calcipotriene 0.005%/betamethasone dipropionate 0.064% once daily to psoriasis plaques on one side of the body and betamethasone dipropionate 0.05% lotion twice daily to plaques on the other side. Biopsies were performed at baseline and after four weeks of treatment to evaluate for epidermal and dermal changes. RESULTS: Treatment with betamethasone lotion resulted in significant decreases in epidermal thickness and dermal thickness. In contrast, treatment with calcipotriene/betamethasone did not lead to significant decreases in epidermal thickness or dermal thickness. Comparing betamethasone and calcipotriene/betamethasone, there was a significantly greater reduction in epidermal thickness with betamethasone lotion versus calcipotriene/betamethasone (P less than .0001). Relative differences in dermal thickness and transepidermal water loss (TEWL) did not reach statistical significance. CONCLUSION: This study is the first to demonstrate that treatment of plaque psoriasis with a combination topical corticosteriod and calcipotriene product results in greater preservation of the skin layers relative to topical corticosteroid use alone. These results hold important ramifications for minimizing cutaneous atrophy in patients receiving treatment with topical corticosteroid .

Angiolymphoid Hyperplasia with Eosinophilia in a Patient With Coccidioidomycosis.

Angiolymphoid hyperplasia with eosinophilia (ALHE) is a rare nodular mass that has not commonly been reported in the literature in association with coccidioidomycosis (CM). Coccidioidomycosis has other known skin manifestations including erythema nodosum and interstitial granulomatous dermatitis. Pulmonary CM is the most common form of the disease and the most common cause of CM-associated rash. This is an important clinical consideration for patients with ALHE who reside in CM-endemic areas, which notably include the southwestern region of the United States, Mexico, and South America. We report the case of an ALHE lesion that resolved following treatment for CM.

Distinct signaling pathways of microtubule inhibitors--vinblastine and Taxol induce JNK-dependent cell death but through AP-1-dependent and AP-1-independent mechanisms, respectively.

Vinblastine and paclitaxel (Taxol) are widely used chemotherapeutic drugs that inhibit the normal function of microtubules causing mitotic arrest and cell death. Despite these similarities, the signaling pathways that mediate and regulate cell death induced by these agents remain incompletely understood. The purpose of this study was to directly compare the two drugs in terms of their ability to activate components of the c-Jun N-terminal protein kinase (JNK) pathway, and to establish the importance of these signaling events in apoptosis induced by these agents. We show that both drugs induce mitotic arrest and subsequent apoptotic cell death with highly similar kinetics and that both activate JNK and induce c-Jun protein and c-jun mRNA expression. Surprisingly, vinblastine induced c-Jun phosphorylation and c-jun transcriptional activation, although Taxol failed to do so. However, inhibition of JNK or an absence of JNK protected against both vinblastine- and Taxol-induced cell death. These results suggest that although JNK activation plays an important role in cell death induced by both agents, vinblastine and Taxol differ markedly with respect to signaling downstream of JNK, with AP-1-dependent and -independent mechanisms, respectively. In addition, these results show, contrary to popular belief, that JNK activation is not necessarily accompanied by c-Jun activation, and thus c-Jun is not an obligate substrate of JNK.

Presenilin-1-dependent transcriptome changes.

Familial forms of Alzheimer's disease (FADs) are caused by the expression of mutant presenilin 1 (PS1) or presenilin 2. Using DNA microarrays, we explored the brain transcription profiles of mice with conditional knock-out of PS1 (cKO PS1) in the forebrain. In parallel, we performed a transcription profiling of the hippocampus and frontal cortex of the FAD-linked DeltaE9 mutant transgenic (TG) mice and matched controls [TG mice expressing wild-type human PS1 (hPS1)]. When the TG and cKO datasets were cross-compared, the majority of the 30 common expression alterations were in opposite direction, suggesting that the FAD-linked PS1 variant produces transcriptome changes primarily by gain of aberrant function. Our microarray studies also revealed an unanticipated inverse correlation of transcript levels between the brains of mice that coexpress DeltaE9 hPS1+ amyloid precursor protein (APP)695 Swe and DeltaE9 hPS1 single transgenic mice. The opposite directionality of these changes in transcript levels must be a function of APP and/or APP derivatives.

9p21 gene locus in Spitz nevi of older individuals: absence of cytogenetic and immunohistochemical findings associated with malignancy.

The diagnosis of Spitz nevus in an elderly individual is often met with skepticism because the lesion can be difficult to distinguish from melanoma and because the probability of a malignant melanoma is higher in older patients. Recently, increased sensitivity for detection of malignant spitzoid neoplasms using 9p21 fluorescence in situ hybridization (FISH) has been described. In this study, we address the question of whether histopathologically typical Spitz nevi occurring in patients 50 years and older show any abnormalities regarding the 9p21 CDKN2A tumor suppressor gene locus. p16 immunohistochemistry (IHC), as well as dual-color FISH for assessment of diploid or hypodiploid status at 9p21, was performed in 25 classic Spitz nevi from patients 50 years and older and was compared with findings in a younger control population. All cases of typical Spitz nevi occurring in older patients retained p16 expression by immunohistochemistry and showed normal, diploid 9p21 FISH signals. Heterozygous loss of 9p21 by FISH was noted in a control case of a 9-year-old girl and is of unknown significance. These findings indicate that p16 expression by immunohistochemistry in classic Spitz nevi correlates well with absence of malignancy-associated cytogenetic abnormalities at 9p21 by FISH independent of the patient's age. Assessment of p16 expression by standard immunohistochemistry may therefore be reassuring in routine clinical practice when the patient is of advanced age, and can be helpful as a screening tool to select IHC-negative cases for extended FISH analysis targeting the 9p21 gene locus.

Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis by phosphorylation of Mcl-1, promoting its degradation and freeing Bak from sequestration.

Mcl-1 is one of the major anti-apoptotic members of the Bcl-2 family of apoptotic regulatory proteins. In this study we investigated the role of Mcl-1 in mitotic arrest-induced apoptosis. Vinblastine treatment of KB-3 cells initially resulted in a phosphatase-sensitive mobility shift in Mcl-1 and then subsequent loss of Mcl-1 protein expression which was prevented by MG132, suggesting that phosphorylation triggered proteosome-mediated degradation. Mcl-1 phosphorylation/degradation was a specific response to microtubule inhibition and did not occur in response to lethal concentrations of DNA damaging agents. Vinblastine treatment caused degradation of Mcl-1 in cells in which apoptosis was blocked by Bcl-xL overexpression, indicating that Mcl-1 degradation was not a consequence of apoptosis. A partial reversible phosphorylation of Mcl-1 was observed in synchronized cells traversing mitosis, whereas more extensive phosphorylation and subsequent degradation of Mcl-1 was observed if synchronized cells were treated with vinblastine. Mcl-1 phosphorylation closely paralleled cyclin B expression, and specific cyclin-dependent kinase (Cdk) inhibitors blocked vinblastine-induced Mcl-1 phosphorylation, its subsequent degradation, and improved cell viability after mitotic arrest. Co-immunoprecipitation studies indicated that Mcl-1 was complexed with Bak, but not Bax or Noxa, in untreated cells, and that Bak became activated in concert with loss of Mcl-1 expression. These results suggest that Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis via Mcl-1 phosphorylation, promoting its degradation and subsequently releasing Bak from sequestration.

Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis.

Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-x(L) and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-x(L)/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-x(L) and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-x(L) mutant but not by a phosphomimetic Bcl-x(L) mutant, confirming Bcl-x(L) as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x(L)/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.

Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 --> Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 --> Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.

True and false discovery in DNA microarray experiments: transcriptome changes in the hippocampus of presenilin 1 mutant mice.

In transcriptome profiling experiments using DNA microarrays, it is critical to maximize putatively true data discovery while keeping the false discovery rate at acceptable levels. Using previously published and verified transcriptome datasets of mice with genetically altered PS1 physiology, we present a simple, robust, and system-specific assessment of type I and type II errors in two independent microarray experimental series. We provide evidence to suggest that for maximizing true discovery and minimizing false discovery, statistical criteria alone are inferior to statistical significance plus magnitude of change criteria. Furthermore, we found that, regardless of the exact criteria used for determining differential expression, different data extraction protocols give rise to different discovery and false discovery rates. In addition, a large proportion of expression differences were both dataset and analytical approach dependent. The data assessment methods presented and discussed in this manuscript can be easily carried out on any microarray dataset using basic spreadsheet functions as the only tool needed. Finally, we provide an in-depth analysis of the hippocampal transcriptome of DeltaE9 hPS1 transgenic mice and mice with a conditional ablation of the PS1 gene.

Transcriptome differences between the frontal cortex and hippocampus of wild-type and humanized presenilin-1 transgenic mice.

OBJECTIVE: The authors investigated the differences between the frontal cortical (Fc) and hippocampal (Hc) transcriptomes of wild type (wt mPS1), humanized presenilin-1 (PS1 [wt hPS1]) and Alzheimer-disease (AD)-linked DeltaE9 hPS1 mutant mice. METHODS: Using high-density oligonucleotide arrays, they recently performed transcriptome profiling of wt mPS1, wt hPS1, and DeltaE9 hPS1 mutant mice. Whereas these studies analyzed the commonalities of gene expression patterns and commonly-regulated genes across the two brain areas and across the animal models, the current study focused on the gene-expression differences across Fc and Hc, two critical AD-affected brain regions. RESULTS: The data revealed that in the wild-type mice, there are significant transcriptome differences between the Fc and the Hc tissue, and these expression differences are maintained in humanized transgenic mice carrying the wt hPS1 gene or DeltaE9 hPS1 mutation. Also, they provide evidence that a subset of genes show disturbed regional Fc-Hc gene-expression ratios in the transgenic mice carrying the DeltaE9 hPS1 mutation. Some of these genes, including stearoyl-Coenzyme A desaturase-2 (Scd2) and Prostaglandin D2 synthase (Ptgds), have been previously implicated in the pathology of AD. CONCLUSIONS: Data suggest that disturbed gene-expression ratios between cortical regions may be an important event in altered brain physiology.