Use of Immunostaining for the diagnosis of Lymphovascular invasion in superficial Barrett's esophageal adenocarcinoma.

BACKGROUND: The prevalence of Barrett's esophageal adenocarcinoma (BEA) is increasing in Japan. Accurate assessment of lymphovascular invasion (LVI) after endoscopic resection or surgery is essential in evaluating treatment response. This study aimed to assess the usefulness of immunostaining in determining the extent of LVI in superficial BEA. METHODS: We retrospectively included 41 patients who underwent endoscopic resection or surgery between January 2007 and July 2018. In all cases, 3-µm serial sections from paraffin-embedded resected specimens were used for hematoxylin and eosin (H-E) staining and immunostaining for D2-40 and CD31. Two specialized gastrointestinal pathologists (T.Y. and T.T.), blinded to clinical information, independently evaluated the extent of LVI from these specimens. The LVI-positivity rate was evaluated with respect to the depth of invasion, changes in the positivity rate on immunostaining, pathological characteristics of patients with LVI, lymph node metastasis or relapse, and course after treatment. RESULTS: H-E staining alone identified LVI in 7 patients (positivity rate: 17.1%). Depths of invasion were categorized based on extension to the submucosa (SM) or deeper. On immunostaining for D2-40 and CD31, additional positivity was detected in 2 patients with SM1 and 1 SM3, respectively; LVI was detected in 10 patients (positivity rate: 24.4%). LVI-positivity rates with invasion of the superficial muscularis mucosa (SMM)/lamina propria mucosa (LPM)/deep muscularis mucosa (DMM), SM 1, 2, and 3 were 0, 75, 28.6, and 55.6%, respectively. CONCLUSIONS: Combined H-E staining and immunostaining is useful in diagnosing LVI in superficial BEA, particularly in endoscopically resected specimens.

Assessment of the Higher-Order Structure of Formulated Monoclonal Antibody Therapeutics by 2D Methyl Correlated NMR and Principal Component Analysis.

Characterization of the higher-order structure (HOS) of protein therapeutics, and in particular of monoclonal antibodies, by 2D 1 H-13 C methyl correlated NMR has been demonstrated as precise and robust. Such characterization can be greatly enhanced when collections of spectra are analyzed using multivariate approaches such as principal component analysis (PCA), allowing for the detection and identification of small structural differences in drug substance that may otherwise fall below the limit of detection of conventional spectral analysis. A major limitation to this approach is the presence of aliphatic signals from formulation or excipient components, which result in spectral interference with the protein signal of interest; however, the recently described Selective Excipient Reduction and Removal (SIERRA) filter greatly reduces this issue. Here we will outline how basic 2D 1 H-13 C methyl-correlated NMR may be combined with the SIERRA approach to collect 'clean' NMR spectra of formulated monoclonal antibody therapeutics (i.e., drug substance spectra free of interfering component signals), and how series of such spectra may be used for HOS characterization by direct PCA of the series spectral matrix. © 2020 U.S. Government. Basic Protocol 1: NMR data acquisition Basic Protocol 2: Full spectral matrix data processing and analysis Support Protocol: Data visualization and cluster analysis.

Optimization and qualification of a functional anti-drug antibody assay for HIV-1 bnAbs.

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function of HIV-1 bnAbs. Using a modified version of the validated TZM-bl HIV-1 neutralization assay, murine anti-idiotype antibodies were utilized to optimize and evaluate parameters of linearity, range, limit of detection, specificity, and precision for measuring inhibitory ADA activity against multiple HIV-1 bnAbs that are in clinical development. We further demonstrate the utility of this assay for detecting naturally occurring ADA responses in non-human primates receiving passive administration of human bnAbs. This functional assay format complements binding-antibody ADA strategies being developed for HIV-1 bnAbs, and when utilized together, will support a multi-tiered approach for ADA testing that is compliant with Good Clinical Laboratory Practice (GCLP) procedures and FDA guidance.

Decreased Expression of Prox1 Is Associated With Postoperative Recurrence in Crohn's Disease.

BACKGROUND: Prox1 is a transcription factor necessary for lymphangiogenesis and lymphatic function. The aim of the study was to assess the correlation between the expression of Prox1 and postoperative recurrence in Crohn's disease [CD]. METHODS: Forty CD patients who underwent ileo-colonic resection were included. Expression levels of Prox1 and D2-40 were detected using immunohistochemistry. Expression levels of Prox1, VEGFR3, and VEGFC protein were also detected in fresh CD specimens using western blotting and quantitative polymerase chain reaction [Q-PCR]. Endoscopic recurrence was used as the endpoint. Patients comprised two groups: endoscopic recurrence [Group R+] and no endoscopic recurrence [Group R-]. RESULTS: Prox1 protein expression was significantly higher in CD than in normal tissues [p <0.05], as detected using both immunohistochemistry and western blotting. Analysis of inter-relationships revealed significant correlation between Prox1 expression and lymphatic vessel density [p <0.001, r = 0.823]. There was also significant correlation between Prox1 expression and the visceral fat area [VFA] [p = 0.002, r = -0.469]. The Group R- patients had significantly higher Prox1 expression than the Group R+ patients [21.08 ± 1.61 versus 15.64 ± 1.17, p = 0.011]. Also, the lymphatic vessel density value was lower in Group R+ than in Group R- patients [6.02 ± 0.39 versus 8.13 ± 0.59, p = 0.004]. Moreover, there was a significant difference in the VFA between Group R- and Group R+ patients [64.43 ± 7.76 versus 90.44 ± 6.11, p = 0.016]. In addition to Prox1, VEGFC/VEGFR3 was found to increase, which was further confirmed using Q-PCR. CONCLUSIONS: Prox1 expression could be useful as a protective factor against recurrence in CD patients. The therapeutic role of Prox1 may lead to improved treatments.

Understanding patterns of invasion: a novel approach to assessment of podoplanin expression in the prediction of lymph node metastasis in oral squamous cell carcinoma.

AIMS: Cellular motility is considered to be central to the process of metastasis, and podoplanin expression can be explored as a prospective link, owing to its ability to modulate the actin cytoskeleton. We aimed to evaluate the tumoral expression of D2-40 (monoclonal antibody against podoplanin) in pathologically neck-node-negative/positive cases (pN0/N+) to characterise the pattern of invasion, potentially explaining the role of various patterns of invasion in causing tumour metastasis. METHODS AND RESULTS: Paraffin-embedded tissue blocks of 60 oral squamous cell carcinoma cases of known nodal status were selected for immunohistochemical staining of tumour (invasive front) by D2-40 along with routine staining by haematoxylin and eosin. Various staining patterns were assessed and evaluated for D2-40 expression, and correlated with nodal status. Tumoral D2-40 expression correspondingly increased with nodal metastasis (P = 0.261). Furthermore, D2-40 staining was more efficient in detecting individual tumour cells, and also characterised the motility factor irrespective of the pattern of invasion (P = 0.001). The pattern of D2-40 staining did not show a significant association with tumour grade, indicating that motility is an overlooked, albeit important, component of the pattern of invasion in routine histological grading. CONCLUSIONS: D2-40 expression successfully identifies the motility profile of tumour, irrespective of the pattern of invasion. The presence of larger motile islands in the tumour cohort supports the concept of 'collective cell migration'. Podoplanin also aids evasion of immune responses by inducing platelet aggregation over tumour cells, thereby favouring distant metastasis. A multivariate model using immunohistochemical staining with D2-40 provides greater sensitivity for the prediction of lymph node metastasis.

Prognostic significance of lymphatic vessel invasion diagnosed by D2-40 in Chinese invasive breast cancers.

Lymphatic vessel invasion (LVI) is promising in determining prognosis and treatment strategies, but the application of LVI as a histopathological criterion in breast cancer patients especially those of different subgroups is controversial. This research aims to evaluate the prognostic value of LVI assessed by D2-40 not only in patients with early invasive breast cancer but also in lymph node-negative, lymph node-positive, luminal A-like, luminal B-like, HER2-enriched, and triple-negative subgroups.The study cohort included 255 patients with a median follow-up of 101 months. Immunohistochemical staining for D2-40 was performed to identify LVI.LVI was present in 64 (25.1%), 15 (12.1%), 49 (37.4%), 19 (20.9%), 23 (27.7%), 13 (31.7%), and 9 (22.5%), respectively, in the whole cohort, lymph node-negative, lymph node-positive, luminal A-like, luminal B-like, HER2-enriched, and triple-negative patients. LVI was associated with large tumor size (P = .04), high histological grade (P = .004), involved lymph node (P < .001), and high expression of Ki-67 (P = .003). No significant difference was found among patients with different subtypes and LVI status. The presence of LVI was significantly associated with adverse disease-free survival in the whole cohort (P < .001), lymph node-negative (P < .001), lymph node-positive (P < .001), luminal A-like (P < .001), and luminal B-like patients (P < .001) in both of the univariate and multivariate survival analysis.This study indicated that the presence of LVI stained by D2-40 provided independent prognostic information not only in the whole cohort but also in the subgroup of patients with lymph node-negative, lymph node-positive, luminal A-like, and luminal B-like diseases, which may make a case for routine clinical assessment of LVI using D2-40.

Extended stability of the rituximab biosimilar CT-P10 in its opened vials and after dilution and storage in polyolefin bags.

The stability of the rituximab biosimilar CT-P10, in 50mL vials at a concentration of 10mg/mL, and after dilution to final concentrations of 1 and 4mg/mL and storage in polyolefin bags at 4°C and 25°C was studied by several orthogonal and complementary methods. No significant change (as defined by a magnitude greater than the inter-batch variability) was observed, for each of the parameters characterizing physical and chemical stability studied, for the two concentrations and temperatures tested, or for any of the three batches tested. This implies that cold-chain rupture and exposure to room temperature up to 15 days both for vials and diluted bags have no deleterious consequence on the quality of the product. Moreover, this extended stability permits safe in-advance preparation, dose-banding or flat-dose, that to avoid unnecessary delays in the management of the patient, improvement of the pharmacy and nurse workload and money saving by avoiding non justified losses of this expensive drug.

El factor de crecimiento endotelial vascular (VEGF) y el fragmento N-terminal de la proteína relacionada con la parathormona (PTHrP) regulan la proliferación de células madre mesenquimales humanas / Vascular endothelial growth factor (VEGF) and the N-terminal portion of parathyroid hormone-related protein (PTHrP) regulate the proliferation of human mesenchymal stem cells

El tejido adiposo contiene un gran número de células madre mesenquimales (Adipose Stem Cells, ASCs) que residen en su estroma vascular. Aunque existe controversia acerca de la capacidad de generar tejido óseo de estas células in vivo, in vitro constituyen un buen modelo de diferenciación osteogénica debido a su semejanza fenotípica con las células estromales de la médula ósea (Bone Marrow Stromal Cells, BMSCs) en cultivo. La diferenciación de las poblaciones osteoprogenitoras de la médula ósea está intensamente regulada por factores locales, como el factor de crecimiento endotelial vascular (VEGF) y la proteína relacionada con la parathormona (PTHrP), que modulan la proliferación de estas poblaciones en distintos estadios de diferenciación. Tanto el VEGF como el fragmento N-terminal de la PTHrP ejercen efectos osteogénicos. En este estudio hipotetizamos que sus efectos sobre la proliferación celular de los osteoprogenitores son dependientes del estadio de diferenciación osteoblástica. Tras confirmar su capacidad de diferenciación in vitro por expresión génica de Runx2 y acumulación de calcio, se analizó la respuesta proliferativa a estímulos con VEGF o PTHrP(1-36) de ASCs sometidas o no a inducción osteogénica. VEGF pero no PTHrP(1-36) estimuló la capacidad proliferativa de las ASCs no inducidas mientras que PTHrP(1-36), pero no VEGF, estimuló la proliferación de las ASCs inducidas, corroborando el papel diferencial de estos factores de crecimiento en distintos estadios de diferenciación (AU)

Adipose tissue contains a large number of mesenchymal stem cells (ASCs) residing in their vascular stroma. Although there is controversy regarding the ability to generate bone tissue from these cells in vivo, the in vitro cells offer a good model of osteogenic differentiation due to its phenotypic similarity with the bone marrow stromal cells (BMSCs) in culture. The differentiation of osteo-progenitor populations of bone marrow is intensely regulated by local factors, such as vascular endothelial growth factor (VEGF) and parathyroid hormone-related protein (PTHrP), which modulate these populations' proliferation in different stages of differentiation. Both the VEGF and the N-terminal fragment of the PTHrP exert osteogenic effects. In this study, we posited that its effects on proliferation of osteo-progenitors are stage dependent of osteoblastic differentiation. After confirming its capacity to in vitro differentiation by Runx2 gene expression and accumulation of calcium, the proliferative response to stimuli was analyzed with VEGF or PTHrP (1-36) of ASCs submitted or not to osteogenic induction. VEGF, but not PTHrP (1- 36), stimulated the proliferative capacity of uninduced ASCs, whereas BMSCs, but not VEGF, stimulated the proliferation of induced ASCs, corroborating the differential role of this growth in different stages of differentiation (AU)

Specific detection and quantitation of bovine IgG in bioreactor derived mouse mAb preparations.

Monoclonal antibody and recombinant protein production benefits greatly from bovine serum as an additive. The caveat is that bovine serum IgG, co-purifies with mAbs and IgG Fc-containing fusion proteins and it presents a contaminant in the end products. In order to analytically validate the products, species specific reagents are needed that react with bovine IgG exclusively. Our attempts to find such commercially available reagents failed. Here, we report the production of species specific mAbs which recognize bovine IgG even in the presence of excess amount of mouse IgG. We present five mAbs: Bsi4028, Bsi4032, Bsi4033, Bsi4034 and Bsi4035 suitable to determine the presence of bovine IgG contamination via ELISA or immunoblotting in bioreactor derived mouse mAb preparations. To quantitate bovine IgG content we developed sensitive sandwich ELISAs capable to detect bovine IgG contaminant in the ng/ml (~10-11M/l) range. Finally, we show that bovine IgG is efficiently removed from bioreactor produced mouse mAb preparation via affinity depletion columns prepared with Bsi4028, Bsi4032, Bsi4033, Bsi4034, Bsi4035 mAbs.

Dosimetric analysis of 177Lu-DOTA-rituximab in patients with relapsed/refractory non-Hodgkin's lymphoma.

OBJECTIVE: Radioimmunotherapy targeting CD20 receptors in lymphoma using radiolabeled chimeric antibodies may lead to better therapeutic responses than cold anti-CD20 antibodies. This study aimed to assess the biodistribution and present reasonable estimates of normal organ doses, including red marrow using Lu-DOTA-rituximab. MATERIALS AND METHODS: Patients with relapsed/refractory CD20+ B-cell non-Hodgkin's lymphoma were recruited into this prospective study. In-house labeling of Lu-DOTA-rituximab was performed and administered after quality assurance. Rituximab (375 mg/m), followed by 50 mCi (1850 MBq) of Lu-DOTA-rituximab was administered as a slow intravenous infusion and emission images were acquired. Regions of interest were drawn for kidney, liver, heart, bladder, spleen, and tumor lesions on both anterior and posterior images. Internal dose estimation was performed using OLINDA v1.0 software. RESULTS: The mean age of the 10 patients (eight men and two women) was 52±13 years. The uptake of radiolabeled antibody was visualized within 30 min of administration in the liver, kidneys, heart, spleen, and bladder. The coefficient of determination (R) was greater than 0.95 for organs and the whole body in all patients. The effective half-life of radioimmunoconjugate was 100±28 h (42-126 h). The critical organ in our study was the red marrow. The average total body dose, effective dose, and effective dose equivalent calculated in all 10 patients were 0.13±0.02, 0.15±0.03, and 0.22±0.04 mGy/MBq, respectively. CONCLUSION: There may be considerable interindividual differences in absorbed doses of organs and generalization or extrapolation of doses in the clinical setting at present is not feasible with Lu-DOTA-rituximab in non-Hodgkin's lymphoma patients. Patient-specific dosimetry is thus recommended to eliminate the variations and reduce the possibility of dose-limiting toxicity.

The Use of Anti-Keratin 903 Antibodies to Visualize Colloid Bodies and Diagnose Lichen Planopilaris.

Cytokeratins are a major component of colloid bodies that are essentially diagnostic of lichen planopilaris (LPP). Here, the authors assess the ability of the cytokeratin 903 antibody (CK-903) to stain colloid bodies and differentiate LPP from other histologically similar appearing primary cicatricial alopecias. A retrospective review of all specimens submitted to the dermatopathology department over a 2-year window identified 18 cases of LPP and 20 cases of histologically similar appearing entities (discoid lupus erythematosus or central centrifugal cicatricial alopecia) through a combination of H&E, elastic van gieson, and periodic acid-schiff stains. All 38 samples were then prospectively stained with CK-903. Colloid bodies were identifiable in 3 of the 18 LPP cases based on H&E alone but were seen in 9 of 18 cases when CK-903 was used. There were no cases where colloid bodies were seen on H&E but not subsequently identified with CK-903. Additionally, there was no CK-903 staining in any of the 20 cases of similar appearing entities except 1 case of discoid lupus erythematosus, which is known to occasionally show colloid bodies. The authors conclude that CK-903 is a useful adjunctive tool that will allow for a quicker, less costly, and more accurate diagnosis of LPP given its ability identify colloid bodies even in the setting of significant inflammation and fibrosis and its advantages over direct immunofluorescence of low cost, short preparation time, and lack of need for a specialized fluorescent microscope.

Hobnail Hemangioma (Superficial Hemosiderotic Lymphovascular Malformation) in Children: A Series of 6 Pediatric Cases and Review of the Literature.

BACKGROUND: Hobnail hemangioma is a small benign vascular malformation of the superficial and mid-dermis with variable clinical presentation. OBJECTIVES: To review the clinical characteristics of hobnail hemangioma in pediatric patients. METHODS: A retrospective chart review performed of all histopathologically confirmed cases of hobnail hemangioma from May 2000 to December 2014. Data on demographics, clinical characteristics, and results of immunohistochemical staining were collected. RESULTS: Four male and 2 female patients identified. Congenital lesions were reported in 3 cases. The most common anatomic location was the extremities. Treatment options included observation and surgical excision. CONCLUSIONS: Hobnail hemangioma is an uncommon benign vascular malformation. Due to its benign nature, treatment is not required. If treatment is indicated, complete surgical excision appears to be the most effective option.

Dual use of E-cadherin and D2-40 immunostaining in unusual meningioma subtypes.

OBJECTIVES: Meningiomas usually can be readily diagnosed on H&E alone, although occasionally immunohistochemistry (IHC) confirmation is desirable. Studies exploring the diagnostic utility of either podoplanin (D2-40) or E-cadherin IHC in meningiomas have conflicted, and no studies exist in which the two IHCs have been used in combination for diagnosis. METHODS: E-cadherin and D2-40 IHC was performed on 77 meningiomas (31 ordinary; eight microcystic; four rare myxoid; six metaplastic; six invasive of orbit, muscle, and/or soft tissue; two metastatic; six brain-invasive World Health Organization [WHO] grade II, nine non-brain-invasive WHO grade II; and five anaplastic WHO grade III), with semi-quantitative scoring on a three-tier scale (0, focal [1+], strong/diffuse [2+]). RESULTS: All meningiomas were either E-cadherin or D2-40 IHC+, with 69 of 77 showing dual immunostaining, most at the 2+ level. No downregulation of E-cadherin IHC was found in invasive or high-grade meningiomas. CONCLUSIONS: Dual E-cadherin/D2-40 IHC can supplement diagnosis of meningioma.

Cluster-based comparison of the peptide mass fingerprint obtained by MALDI-TOF mass spectrometry. A case study: long-term stability of rituximab.

We evaluated the use of the peptide mass fingerprint (PMF) obtained by matrix assisted laser desorption and ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) to track changes in the structure of a protein. The first problem we had to overcome was the inherent complexity of the PMF, which makes it difficult to compare. We dealt with this problem by developing a cluster-based comparison algorithm which takes into account the proportional error made by the mass spectrometer. This procedure involves grouping together similar masses in an intelligent manner, so that we can determine which data correspond to the same peptide (any slight differences can be explained as experimental errors), and which of them are too different and thus more likely to represent different peptides. The proposed algorithm was applied to track changes in a commercially available monoclonal antibody (mAb), namely rituximab (RTX), prepared under the usual hospital conditions and stored refrigerated (4 °C) and frozen (-20 °C) for a long term study. PMFs were obtained periodically over three months. For each checked time, five replicates of the PMFs were obtained in order to evaluate the similarities between them by means of the occurrences of the particular peptides (m/z). After applying the algorithm to the PMF, different approaches were used to analyse the results. Surprisingly, all of them suggested that there were no differences between the two storage conditions tested, i.e. the RTX samples were almost equally well preserved when stored refrigerated at 4 °C or frozen at -20 °C. The cluster-based methodology is new in protein mass spectrometry and could be useful as an easy test for major changes in proteins and biopharmaceutics for diverse applications in industry and other fields, and could provide additional stability data in relation to the practical use of anticancer drugs.

Expression of lymphatic markers and lymphatic growth factors in psoriasis before and after anti-TNF treatment

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results. .

Expression of lymphatic markers and lymphatic growth factors in psoriasis before and after anti-TNF treatment.

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results.

An immunohistochemical analysis of a newly developed, mouse monoclonal p40 (BC28) antibody in lung, bladder, skin, breast, prostate, and head and neck cancers.

CONTEXT: Immunohistochemistry is important to the pathologist for accurate diagnosis of lung cancer. In recent studies, a rabbit polyclonal p40 (RPp40) antibody demonstrated equivalent staining versus anti-p63 in lung squamous cell carcinoma, and superior specificity because it stains a lesser percentage of lung adenocarcinoma. OBJECTIVES: To develop an anti-p40 mouse monoclonal antibody (MMp40) for immunohistochemistry, and to evaluate its sensitivity and specificity in normal and neoplastic tissues, with emphasis on lung cancer. DESIGN: The MMp40 (BC28) antibody was developed and tested for specificity and sensitivity on normal (n = 34) and neoplastic tissues (n = 493). Staining of MMp40, p63, and RPp40 were directly compared in lung cancers, and MMp40 was evaluated in breast, bladder, skin, prostate, and head and neck cancers. Benign prostate glands and prostatic intraepithelial neoplasia were also tested in a direct comparison of MMp40 versus p63. RESULTS: The MMp40 provided equivalent staining versus RPp40 and p63 in lung squamous cell carcinoma, but stained a lesser percentage of lung adenocarcinoma than p63 did. The MMp40 staining was observed in a greater percentage of urothelial carcinomas, squamous and basal cell skin cancers, and head and neck cancers of squamous cell origin. No breast-infiltrating ductal carcinomas or prostatic adenocarcinomas were stained. The MMp40 expression in basal cells of prostate glands and prostatic intraepithelial neoplasia were almost identical to those of p63. CONCLUSION: The MMp40 (BC28) monoclonal antibody is a high-quality screening antibody for determining squamous cell carcinoma in lung cancers, skin cancers of squamous or basal cell origin, squamous cell head and neck cancers, and urothelial carcinomas.

Use of automated image analysis in evaluation of Mesothelioma Tissue Microarray (TMA) from National Mesothelioma Virtual Bank.

The National Mesothelioma Virtual Bank (NMVB) was established to provide annotated biospecimens to the mesothelioma research community. The resource provides tissue microarrays (TMA) to evaluate the biomarkers along with a variety of other resected mesothelioma specimens. In this manuscript, we describe the immunohistochemical evaluation of the mesothelioma TMA with three key antibodies that are used in making the diagnosis of mesothelioma, and compared the immunohistochemical assessment between manual scoring and image analysis. The TMA was assessed for the immunohistochemical expression of calretinin (N=39), cytokeratin (CK) 5/6 (N=33), and D2-40 (N=37). Immunohistochemistry was evaluated by semi-quantitative (manual) scoring using light microscope (MS) and by automated image analysis (AS). Calretinin staining was seen in both cytoplasmic and nuclear locations. CK5/6 stain was localized to the cytoplasm. D2-40 stain showed only membranous expression in our cases. • Based on the pathologist's scores, calretinin was positive in 31 of the 39 cases (80%), CK 5/6 in 15 of the 33 cases (46%) and D2-40 in 18 of the 37 cases (49%). • The percent-positive agreement between manual scores and image analysis was 90% (35/39), 94% (31/33), and 95% (35/37) for calretinin, CK 5/6, and D2-40, respectively. There was a substantial agree-ment between manual and automated scores for calretinin (kappa = 0.614) and an almost perfect agreement for CK5/6 (kappa = 0.879) and D2-40 (kappa = 0.892). Our study confirms that the immunohistochemical staining pattern of mesotheliomas in the NMVB UPMC TMA is similar to other studies. Our findings also show that automated image analysis provides similar results to manual scoring by pathologists, and provides a reproducible, objective, and accurate platform for immunohistochemical assessment of biomarker expression.

Diffuse dermal angiomatosis of the breast: clinical and histopathological features.

INTRODUCTION: Diffuse dermal angiomatosis is an entity in the spectrum of reactive angiomatoses characterized by erythematous plaques that mainly affect the lower extremities of patients with a personal history of peripheral vascular disease. Involvement of the breast is a rare event that has only been described as single cases in women with large breasts. OBJECTIVE: Our main aim is to report three rare cases of diffuse dermal angiomatosis of the breast and to better define their clinical, histopathological, and immunohistochemical characteristics. Comorbidities and management will also be discussed. METHODS: A retrospective search of patients with the diagnosis of diffuse dermal angiomatosis of the breast was made. Databases of three hospitals, Hospital 12 de Octubre (Madrid, Spain), Hospital La Fe (Valencia, Spain), and Clinica Dermatologica, University of Genoa (Italy), were included in the analysis. RESULTS: Three middle-aged women who were heavy smokers were found. Physical examination revealed several livedoid plaques on both breasts. Painful ulceration over the violaceous lesions was observed in two cases. Histological features included a diffuse proliferation of spindle-shaped endothelial cells with focal small vessel formation occupying the full thickness of the dermis with scanty extravasated erythrocytes, showing positivity for CD31, CD34, and SMA-α and negativity for D2-40. Management was focused on a strict control of comorbidities, especially on the cessation of smoking habit that led to a conspicuous improvement in all patients. CONCLUSIONS: We describe all those clinicopathologic features that define diffuse dermal angiomatosis of the breast, which should be considered a distinctive variant into the group of cutaneous angiomatoses. In our experience, a strict control of smoking habit must be the first step in the management of this entity.