A Rare Case of Prurigo Pigmentosa in a Danish Sibling Couple.

Prurigo pigmentosa (PP) is probably underdiagnosed due to lack of awareness. Previously, it was assumed that PP primarily affected Japanese females; however, more cases are reported worldwide, and the pathogenesis is still not completely understood. In this case report, we present two healthy Danish siblings, who developed PP approximately 2 weeks after starting a ketogenic diet, suggesting that both increased levels of ketone bodies in the blood together with a genetic predisposition might play a role in the development of PP.

Eosinophilic pustular folliculitis of infancy: A rare diagnosis in children.

Remember EPFI as a differential diagnosis in children with a rash on the scalp and no effect of antibiotic treatment.

Ki-67 Proliferation Index in Breast Cancer as a Function of Assessment Method: A NordiQC Experience.

Immunohistochemical staining for Ki-67 is used to calculate a Ki-67 proliferation index (PI) that carries prognostic and predictive information in various cancers including breast carcinomas. Studies have documented challenges for observers to reproducibly estimate the Ki-67 PI. At present, no international consensus exists concerning scoring method (eg, hotspots vs. overall average, digital vs. manual counting) or even the definition of a Ki-67-positive cell. To clarify the approach to Ki-67 scoring and evaluation of the interobserver agreement among participants in the Nordic Immunohistochemical Quality Control (NordiQC) Breast Cancer Module, a study was set up on the basis of an online web module containing 15 digitized tissue microarray cores of breast carcinomas stained for Ki-67 in the NordiQC reference laboratory. All participants were invited to attend the study. In addition to Ki-67 scoring, they were asked to disclose their preferred method for Ki-67 estimation and their job title. For comparison, slides were analyzed using a Digital Image Analysis algorithm based on Virtual Double Staining. In total, 199 participants enrolled for the study. Overall, there was a good correlation in Ki-67 PIs among the participants, although results for some cores varied significantly. However, when applying a cutoff of 20%, a relatively low κ value of 0.52 was observed. Participants scoring in hotspots reported higher Ki-67 PIs than participants estimating an overall average, and, not surprisingly, participants who considered weak Ki-67 nuclear staining positive obtained higher Ki-67 PIs than those who did not. Ki-67 PI was not correlated to job title. The Virtual Double Staining algorithm obtained Ki-67 values close to the mean value of the human observers. Our study underlines the need for international standardization and guidelines in estimation of Ki-67 PI. Digital Image Analysis may be a useful tool in this process.

Impact of Primary Antibody Clone, Format, and Stainer Platform on Ki67 Proliferation Indices in Breast Carcinomas.

Ki67 is a nuclear protein expressed during the active phases of the cell cycle, which makes it a biomarker of cell proliferation. In clinical pathology settings, immunohistochemical (IHC) detection of Ki67 is used to calculate Ki67 proliferation indices (PIs), which have prognostic information and are used to subdivide breast carcinomas and neuroendocrine neoplasias. Calculation of Ki67 PIs is notoriously hard and prone to intraobserver and interobserver variance. In addition, IHC protocol settings [such as primary antibody (Ab) clone, clone format, and stainer platform] can affect the result of the IHC assays and in turn the Ki67 PI. Digital image analysis has been suggested as a useful tool to standardize Ki67 counting. Recently, virtual double staining, a computer algorithm segmenting Ki67 and Ki67 tumor cells using digitally fused parallel cytokeratin and Ki67-stained slides, has been introduced. In this study, we compare Ki67 PIs obtained by virtual double staining in 41 breast carcinomas stained using the most commonly used commercially available primary Ab clones and formats on the main stainer platforms. IHC protocols for the concentrated (conc) Ab and platform combinations were optimized for the highest analytical sensitivity and optimal signal-to-noise ratio, whereas ready-to-use (RTU) formats were used, as recommended by the vendor. Significant differences in the mean Ki67 PIs (relativized to the mean core Ki67) were observed not only between the different Ab clones but also the different formats and stainer platforms; Ki67 PIs with SP6 conc stained on the Ventana BenchMark ULTRA platform were on average 11.9 percentage points (pp) higher than the mean core average, whereas with Ab 30.9 RTU on the Ventana platform, they were 10.4 pp higher. Mib1 RTU (Dako Autostainer Link 48) and MM1 RTU (Leica Bond) provided 8.6 and 12.5 pp lower Ki67 PIs, respectively. Mib1 conc and SP6 conc on the Dako Autostainer and Leica Bond provided similar results-close to the overall average. Significant variations in the proportion of tumors with Ki67 high-level expression (Ki67 PI ≥20%) were observed among Ab, format, and stainer platform combinations. The results underline the challenges in the comparison of Ki67 PIs across Abs, formats, and platforms. Researchers and clinicians need to account for these differences when reporting Ki67 PIs. To advance the usefulness of Ki67 PIs in the research and clinical setting, standardization of Ki67 IHC assays is needed.

Severe Infantile Bullous Pemphigoid Treated with Dapsone after Bridging with Systemic Glucocorticoid.

We present a case of severe and treatment-refractory bullous pemphigoid in a 3-month-old child. After topical and systemic corticoid treatment proved inefficient, dapsone 0.75 mg/kg was added initially without success. Disease control was reached with dapsone 1.5 mg/kg in addition to both topical and systemic glucocorticoid treatment, leaving the child with several side effects of the glucocorticoid treatment.

Immunohistochemical CD271 expression correlates with melanoma progress in a case-control study.

Putative cancer stem cell (CSC) markers have arisen from melanoma mouse and in vitro models, but their expression in paraffin embedded patient samples relative to clinical outcome remains largely unexplored. Rather than cells of the tumour bulk, conceivably, CSC drive tumour progression. Accordingly, complete eradication may prevent melanoma relapse. Because elevated tumour-cell proliferation is an established indicator of aggressive disease, this study aimed to investigate the correlation between melanoma recurrence and proliferation of putative CSC that express CD271, CD166, or CD20. Additionally, the expression of these markers was studied in naevi, melanomas, and their recurrence. In melanoma patients, 30 with relapse (cases) and 30 without (controls) were matched for tumour thickness, ulceration, Clark level, subtype, site, gender, and age. One paraffin-embedded section of the patients' primary melanoma (n = 60), relapse (n = 21), and naevus (n = 17) were immunohistochemically double-stained for Ki-67/MART1 and single-stained for CD271, CD166, and CD20. Their whole slide images were aligned as virtual quadruple stains. Image analysis established proliferation indices of each putative stem cell marker and the tumour bulk in addition to the markers' percentage level in tumour areas and the epidermis. In cases vs controls, median dermal proliferation indices (no./mm2) were 211 vs 103 (p = 0.04) for CD271, 512 vs 227 (p = 0.3) for CD166, 184 vs 97 (p = 0.3) for CD20, and 95 vs 103 (p = 0.6) for the tumour bulk. Of additional interest, epidermal CD271+ keratinocytes totalled 8.8% in naevi and 0.98% in melanomas (p = 0.0007). Even though differences between naevi and melanomas also were observed for CD166 in both the epidermis (p = 0.002) and dermis (p = 0.006), they were visually less apparent. CD20+MART1+ cells were absent in half of the melanomas, and all naevi and relapses. In conclusion, high levels of CD271+Ki-67+MART1+ cells were linked to melanoma relapse as opposed to common Ki-67 indices in this particular case-control study. With further investigation, such cells could be potential targets of therapy. Especially, loss of epidermal CD271+ keratinocytes seemed necessary for melanoma development; hence, identification may serve as a diagnostic tool with additional research.

Telomerase reverse transcriptase promoter mutations and solar elastosis in cutaneous melanoma.

The aims of this study were to assess the prognostic potential of solar elastosis grading and telomerase reverse transcriptase (TERT) promoter mutations (TERTp) in melanoma and to evaluate whether an association between solar elastosis and TERTp exists. Solar elastosis in the dermis was evaluated in hematoxylin and eosin-stained whole slides from 486 malignant melanomas. Pyrosequencing was used to detect TERTp in 189 samples. There was no association between solar elastosis and TERTp (P=0.3). Severe elastosis was associated with older age (P<0.0001), ulceration (P=0.03), and location in the head/neck region (P<0.0001). The absence of elastosis was associated with younger age (P<0.0001), benign nevus remnants (P=0.001), and a positive BRAF V600E expression (P<0.0001). Severe elastosis predicted a worse relapse-free survival (hazard ratio: 2.18; 95% confidence interval: 1.30-3.64; P=0.003). However, it was not independent of age. TERTp was not associated with any adverse prognostic or clinicopathological outcome, nor any mitogen-activated protein kinase-related protein expressions. However, at a cutoff corresponding to the sensitivity of Sanger sequencing, TERTp predicted melanoma-specific death independently of age, and was associated with Breslow thickness, ulceration, tumor stage at diagnosis, BRAF V600E oncoprotein, and absence of p16 expression. In conclusion, TERTp were not related to severe elastosis and may thus be triggered by both chronic and acute intermittent sun exposure, the latter not visible on ordinary hematoxylin and eosin-stained slides. Neither TERTp nor severe elastosis predicted an adverse outcome in melanoma. An absence of elastosis was seen in younger melanoma patients and may be used to select those melanomas originating in a nevus, which often harbors a BRAF mutation.

Quantification of microRNA-21 and microRNA-125b in melanoma tissue.

Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours. Using array analysis, samples mainly clustered according to their biological group (benign vs. malignant) and 77 miRNAs differed significantly between nevi and melanoma samples. Increase of miR-21 and miR-142, and decrease of miR-125b, miR-211, miR-101 and miR-513c in the melanomas were verified in both cohorts using qPCR, whereas the decrease of miR-205 observed with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.

Ibrutinib-Associated Skin Toxicity: A Case of Maculopapular Rash in a 79-Year Old Caucasian Male Patient with Relapsed Waldenstrom's Macroglobulinemia and Review of the Literature.

Waldenstrom's macroglobulinamia (WM) is a rare malignant lymphoproliferative disorder, characterized by monoclonal IgM paraproteinemia and neoplastic proliferation of malignant lymphoplasmacytoid cells in the bone marrow. Traditionally, WM has been treated with modalities similar to those used in the management of other indolent lymphomas. Just recently, based on impressive clinical trial results in heavily pretreated WM patients, a new Bruton Tyrosine Kinase-inhibitor, Ibrutinib, has been approved for the treatment of this disorder. As the use of Ibrutinib in WM outside clinical trials is still limited, only few clinical reports illustrating treatment side effects are currently available. Here we review the current literature specific on Ibrutinib-associated rash in hematologic patients, and report on an elderly patient with WM, who developed a red maculopapular non-pruritic rash 12 weeks after starting Ibrutinib therapy. Without modifications of the ongoing Ibrutinib schedule, the rash regressed within two weeks of treatment with topical steroid-containing dermatological compounds.

Proliferation assessment in breast carcinomas using digital image analysis based on virtual Ki67/cytokeratin double staining.

Manual estimation of Ki67 Proliferation Index (PI) in breast carcinoma classification is labor intensive and prone to intra- and interobserver variation. Standard Digital Image Analysis (DIA) has limitations due to issues with tumor cell identification. Recently, a computer algorithm, DIA based on Virtual Double Staining (VDS), segmenting Ki67-positive and -negative tumor cells using digitally fused parallel cytokeratin (CK) and Ki67-stained slides has been introduced. In this study, we compare VDS with manual stereological counting of Ki67-positive and -negative cells and examine the impact of the physical distance of the parallel slides on the alignment of slides. TMAs, containing 140 cores of consecutively obtained breast carcinomas, were stained for CK and Ki67 using optimized staining protocols. By means of stereological principles, Ki67-positive and -negative cell profiles were counted in sampled areas and used for the estimation of PIs of the whole tissue core. The VDS principle was applied to both the same sampled areas and the whole tissue core. Additionally, five neighboring slides were stained for CK in order to examine the alignment algorithm. Correlation between manual counting and VDS in both sampled areas and whole core was almost perfect (correlation coefficients above 0.97). Bland-Altman plots did not reveal any skewness in any data ranges. There was a good agreement in alignment (>85 %) in neighboring slides, whereas agreement decreased in non-neighboring slides. VDS gave similar results compared with manual counting using stereological principles. Introduction of this method in clinical and research practice may improve accuracy and reproducibility of Ki67 PI.

Automated quantification of proliferation with automated hot-spot selection in phosphohistone H3/MART1 dual-stained stage I/II melanoma.

BACKGROUND: Staging of melanoma includes quantification of a proliferation index, i.e., presumed melanocytic mitoses of H&E stains are counted manually in hot spots. Yet, its reproducibility and prognostic impact increases by immunohistochemical dual staining for phosphohistone H3 (PHH3) and MART1, which also may enable fully automated quantification by image analysis. To ensure manageable workloads and repeatable measurements in modern pathology, the study aimed to present an automated quantification of proliferation with automated hot-spot selection in PHH3/MART1-stained melanomas. METHODS: Formalin-fixed, paraffin-embedded tissue from 153 consecutive stage I/II melanoma patients was immunohistochemically dual-stained for PHH3 and MART1. Whole slide images were captured, and the number of PHH3/MART1-positive cells was manually and automatically counted in the global tumor area and in a manually and automatically selected hot spot, i.e., a fixed 1-mm(2) square. Bland-Altman plots and hypothesis tests compared manual and automated procedures, and the Cox proportional hazards model established their prognostic impact. RESULTS: The mean difference between manual and automated global counts was 2.9 cells/mm(2) (P = 0.0071) and 0.23 cells per hot spot (P = 0.96) for automated counts in manually and automatically selected hot spots. In 77 % of cases, manual and automated hot spots overlapped. Fully manual hot-spot counts yielded the highest prognostic performance with an adjusted hazard ratio of 5.5 (95 % CI, 1.3-24, P = 0.024) as opposed to 1.3 (95 % CI, 0.61-2.9, P = 0.47) for automated counts with automated hot spots. CONCLUSIONS: The automated index and automated hot-spot selection were highly correlated to their manual counterpart, but altogether their prognostic impact was noticeably reduced. Because correct recognition of only one PHH3/MART1-positive cell seems important, extremely high sensitivity and specificity of the algorithm is required for prognostic purposes. Thus, automated analysis may still aid and improve the pathologists' detection of mitoses in melanoma and possibly other malignancies.

Tumour-associated mast cells in classical Hodgkin's lymphoma: correlation with histological subtype, other tumour-infiltrating inflammatory cell subsets and outcome.

The tumour microenvironment in classical Hodgkin's lymphoma (cHL) is characterised by a minor population of neoplastic Hodgkin and Reed-Sternberg cells within a heterogeneous background of non-neoplastic bystanders cells, including mast cells. The number of infiltrating mast cells in cHL has been reported to correlate with poor prognosis. We used immunohistochemistry to assess the degree of tumour-infiltrating mast cells in cHL tissue microarrays and correlated this with clinico-pathological features and prognosis in a cohort of homogeneously treated patients with Hodgkin's disease. A high degree of tumour mast cells was associated with nodular sclerosis (NS) subtype histology (P = 0.0002). Moreover, the number of mast cells was inversely correlated with the numbers of CD68+ and CD163+ macrophages (P = 0.0001 and P = 0.003, respectively) and with the number of granzyme+ cytotoxic cells (P = 0.004). The degree of mast cell infiltration was not a prognostic factor in cHL of nodular sclerosis subtype. In contrast, in mixed cellularity cHL a high number of intratumoral mast cells correlated with significantly poorer outcome both in terms of overall (P = 0.03) and event-free survival (P = 0.01). Further studies are warranted into the biological mechanisms underlying this adverse outcome and their possible therapeutic implications.

KIT is a frequent target for epigenetic silencing in cutaneous melanoma.

The receptor tyrosine kinase KIT and its ligand, stem cell factor (SCF), are essential for the proliferation and survival of normal melanocytes. In melanomas arising on mucosal, acral, and chronically sun-damaged skin, activating KIT mutations have been identified as oncogenic drivers and potent therapeutic targets. Through an initial whole-genome screen for aberrant promoter methylation in melanoma, we identified the KIT promoter as a target for hypermethylation in 43/110 melanoma cell lines, and in 3/12 primary and 11/29 metastatic cutaneous melanomas. Methylation density at the KIT promoter correlated inversely with promoter activity in vitro and in vivo, and the expression of KIT was restored after treatment with the demethylating agent 5-aza-2'-deoxycytidine. Hypermethylation of KIT showed no direct or inverse correlations with well-documented melanoma drivers. Growth of melanoma cells in the presence of SCF led to reduced KIT expression and increased methylation density at the KIT promoter, suggesting that SCF may exert a selection pressure for the loss of KIT. The frequent loss of KIT in cutaneous melanoma by promoter hypermethylation suggests that distinct KIT signaling pathways have opposing roles in the pathogenesis of melanoma subtypes.

Digital pathology for the validation of tissue microarrays in peripheral T-cell lymphomas.

Systematic validation of construction and analysis parameters when using tissue microarray (TMA) in rare, morphologically heterogenous entities such as peripheral T-cell lymphoma (PTCL) is not reported. We describe a tissue-saving virtual TMA to predetermine the number of cores needed to represent whole tissue sections (WTS) from the same biopsies, using automated and traditional manual methods for the quantification of immunohistochemical stains. Whole paraffin hematoxylin and eosin- and immunohistochemical (CD2, CD30, and Ki-67)-stained sections from 30 PTCLs were digitalized. A virtual TMA with six 1-mm cores per slide was designed to compare agreements in the immunohistochemical scoring. Using digital image analysis and manual stereological counting, immunohistochemical positivity was quantified. Associations were analyzed using the Bland-Altman and correlation plots. In PTCL, we report that 4 cores are required to represent WTS results (ie, agreement within ±10%). High concordance was demonstrated between digital results obtained with WTS compared with 4-core virtual TMA (correlation coefficients: 0.89-0.98), and in the comparative evaluation of 4-core virtual TMA by digital image analysis versus manual stereology (correlation coefficients: 0.91 to 0.99). Virtual TMAs provide an efficient tool for optimizing and validating TMA construction parameters when planning a study. The method can be applied to the same tissues used in a subsequent formal study, without wasting scarce tissue resources. In PTCL, TMAs constructed with four 1-mm cores are representative of WTS. In parallel tests using TMAs and WTS from PTCLs, there is a high level of agreement comparing automated digital with manual stereological methods for the quantification of immunohistochemical biomarker staining.

Automated quantification of MART1-verified Ki-67 indices: useful diagnostic aid in melanocytic lesions.

The MART1-verified Ki-67 proliferation index is a valuable aid to distinguish melanomas from nevi. Because such indices are quantifiable by image analysis, they may provide a novel automated diagnostic aid. This study aimed to validate the diagnostic performance of automated dermal Ki-67 indices and to explore the diagnostic capability of epidermal Ki-67 in lesions both with and without a dermal component. In addition, we investigated the automated indices' ability to predict sentinel lymph node (SLN) status. Paraffin-embedded tissues from 84 primary cutaneous melanomas (35 with SLN biopsy), 22 melanoma in situ, and 270 nevi were included consecutively. Whole slide images were captured from Ki-67/MART1 double stains, and image analysis computed Ki-67 indices for epidermis and dermis. In lesions with a dermal component, the area under the receiver operating characteristic (ROC) curve was 0.79 (95% confidence interval [CI], 0.72-0.86) for dermal indices. By excluding lesions with few melanocytic cells, this area increased to 0.93 (95% CI, 0.88-0.98). A simultaneous analysis of epidermis and dermis yielded an ROC area of 0.94 (95% CI, 0.91-0.96) for lesions with a dermal component and 0.98 (95% CI, 0.97-1.0) for lesions with a considerable dermal component. For all lesions, the ROC area of the simultaneous analysis was 0.89 (95% CI, 0.85-0.92). SLN-positive patients generally had a higher index than SLN-negative patients (P ≤ .003). Conclusively, an automated diagnostic aid seems feasible in melanocytic pathology. The dermal Ki-67 index was inferior to a combined epidermal and dermal index in diagnosis but valuable for predicting the SLN status of our melanoma patients.

Nodal distribution, stage migration due to diameter measurement and the prognostic significance of metastasis volume in melanoma sentinel lymph nodes: a validation study.

To validate metastasis location, maximum metastasis diameter (MMD) measurement induced stage migration and the prognostic significance of total metastatic volume (TMV) in melanoma sentinel lymph nodes (SLNs). A new 227 patient SLN cohort examined by complete step sectioning (250 µm) was tested for TMV and MMD prognostic significance; metastasis location by three different three-level protocols (Central vs Peripheral vs Global); and potential treatment changing down-staging by two restricted five-level protocols [Reduced Central (250 µm) vs Reduced Even (500 µm) sections]. Both TMV and MMD independently predicted recurrence (TMV, hazard ratio (HR): 1.21; 95% confidence interval (CI) 1.07-1.37; p = 0.003; and MMD, HR: 1.46; 95% CI 1.08-1.98, p = 0.02) and melanoma-specific death (TMV, HR: 1.30; 95% CI 1.11-1.51; p = 0.001; and MMD, HR: 1.82; 95% CI 1.25-2.66, p = 0.002). The Central, Peripheral and Global protocols detected 72%, 76% and 83% of metastases found by complete step sectioning. Based on MMD, using the Reduced Central or Reduced Even protocols, potential treatment changing down-staging occurred in 20 (20%) or 13 (13%) of SLN-positive patients. This validation study establishes that: (i) metastases are globally located in melanoma SLNs; (ii) MMD but not TMV leads to uni-directional stage migration; and (iii) TMV analysis is of prognostic significance.

Immunohistochemical analysis of molecular drivers in melanoma identifies p16 as an independent prognostic biomarker.

AIMS: To perform immunohistochemical (IHC) analysis of molecular drivers related to the development and maintenance of melanoma and to assess their value as diagnostic and prognostic melanoma biomarkers in routine clinical practice. METHODS: Tissue microarrays constructed from a cohort of primary melanomas (n=355), benign naevi (n=37) and melanoma metastases (n=14) were evaluated for IHC expression of c-KIT, BRAF(V600E), MITF, p16, p53 and PTEN, as well as for pERK, a surrogate marker for mitogen-activated protein kinase pathway activation. The results were correlated with clinicopathological parameters and clinical outcome. RESULTS: Absent p16 expression and reduced MITF expression were both associated with the adverse prognostic markers ulceration (p=0.009 and p<0.0001, respectively), advanced tumour stage (p<0.0001 and p=0.001, respectively) and higher Breslow thickness (both p<0.0001), as well as with an adverse overall relapse-free survival (p<0.0001 and p=0.003, respectively). Absence of p16 expression predicted overall relapse-free (p=0.02) and distant metastasis-free (p=0.04) survival, independently of Breslow thickness, ulceration and tumour stage. CONCLUSIONS: IHC determined p16 expression is an independent prognostic biomarker of potential value in routine melanoma diagnostic practice.