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.

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.

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.

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.

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.

Real time analysis of binding between Rituximab (anti-CD20 antibody) and B lymphoma cells.

CD20, expressed on greater than 90% of B-lymphocytic lymphomas, is an attractive target for antibody therapy. Rituximab is a chimeric murine/human-engineered monoclonal antibody which can selectively deplete CD20-expressing cells in peripheral blood and lymphoid tissues. The immobilization of B-lymphoblast-like Burkitt's lymphoma Raji cells on the quartz crystal microbalance (QCM) gold electrode surface using arginine-glycine-aspartic acid (RGD) tripeptide was electrochemically confirmed. The real-time processes of attachment of Raji cells on the gold electrode and the subsequent binding of Rituximab to the cells were studied using a QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (Δf0) in the studied antibody concentration range from 5 to 250 µg mL(-1) following the Langmuir adsorption model. From these observations, the apparent binding constant between a single-layer of Rituximab and Raji cells was calculated to be 1.6 × 10(6) M(-1). Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved the specific interaction between Rituximab and B cells. The effects of Ca(2+) and Mn(2+) ions on the Rituximab-Raji cell interaction were also studied providing the enhanced QCM signals, in particular with Ca(2+), further indicating that CD20 is a calcium ion channel that can transport these metal ions into the cells and accelerate the cell lysis induced by Rituximab. Thus, the real time capability of QCM and its simplicity of operation are shown to be highly suitable for multipurpose studies on living cells including cell-immobilization, cytotoxicity of drugs, and the cell action mechanisms.

Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines.

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reversephase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stabilityindicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm×4.6 mm, 5 µm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ =214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines.We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.

Therapeutic strategies for well-differentiated papillary mesothelioma of the peritoneum.

OBJECTIVE: Well-differentiated papillary mesothelioma is an uncommon subtype of mesothelioma with a frequently indolent course, although it occasionally manifests in a more aggressive form. To establish a treatment strategy for this rare disease, we report the clinical characteristics and outcomes of 15 patients with well-differentiated papillary mesothelioma. METHODS: All pathologically diagnosed well-differentiated papillary mesothelioma cases were reviewed between 1998 and 2012. RESULTS: Of the 15 cases, 8 and 7 presented with single and multiple lesions, respectively. All cases with single lesions were asymptomatic, while 4 out of the 7 cases with multiple lesions were symptomatic. After tumor excision, none of the eight single-lesion cases experienced tumor recurrence. Among the other seven cases with multiple lesions, only one patient with disseminated lesions died due to disease burden. Five patients with multiple lesions received cisplatin-based intravenous or intraperitoneal chemotherapy, with a mix of complete (n= 2) and partial (n= 2) responses observed. Of particular note, one patient receiving cisplatin and pemetrexed combination chemotherapy experienced complete tumor resolution without any serious toxicity. CONCLUSIONS: We recommend different treatment strategies based on the disease status. If the tumor is completely resectable, an excisional biopsy seems to be sufficient. If complete resection is unavailable for the asymptomatic patient with a localized tumor extent, close follow-up is an appropriate option. When the tumor is extensive or accompanied by symptoms, chemotherapy should be strongly considered.

Cyclo-oxygenase-2 expression is associated with vascular endothelial growth factor C expression and lymph node metastasis in oral squamous cell carcinoma.

PURPOSE: Cervical lymph node metastasis in oral squamous cell carcinoma (OSCC) is recognized as a poor prognostic factor, although its mechanism remains unclear. Recently, cyclo-oxygenase-2 (COX-2) level has been found to correlate highly with vascular endothelial growth factor C (VEGF-C) and lymph node metastasis, as in other solid tumors. However, there has been no report of this correlation in OSCC. Therefore, the aim of this study was to investigate whether COX-2 immunohistochemical expression in OSCC was associated with VEGF-C expression, histopathologic parameters, and lymph node metastasis. MATERIALS AND METHODS: Lymphatic vessel density, VEGF-C, and COX-2 immunohistochemical expression were examined pathologically in 60 specimens of invasive OSCC. Relations of histopathologic parameters to lymph node metastasis were analyzed. RESULTS: Expression levels of VEGF-C and COX-2 and lymphatic vessel density in the lymph node metastatic group were significantly higher than in the nonmetastatic group (P < .01). A significant correlation was found between the expression levels of VEGF-C and COX-2 (r = 0.512; P < .001). COX-2 expression was significantly related to lymph node metastasis (P = .004) and VEGF-C expression (P = .005). Univariate analysis showed that survival time was impaired by higher COX-2 and VEGF-C expression levels. Multivariate survival analysis showed that COX-2 expression was an independent prognostic factor. CONCLUSION: This study showed that VEGF-C expression was upregulated by COX-2 in OSCC. High VEGF-C expression appears to promote peritumoral lymphangiogenesis. These data indicated that lymph node metastasis is promoted by COX-2 and VEGF-C in OSCC.

D2-40 immunohistochemical overexpression in cutaneous squamous cell carcinomas: a marker of metastatic risk.

BACKGROUND: Approximately 4% of cutaneous squamous cell carcinomas (cSCCs) develop lymphatic metastases. The value of lymphatic endothelial markers to enhance the detection of lymphatic tumor invasion in cSCC has not been assessed previously. OBJECTIVE: We sought to evaluate the use of the antibody D2-40, a podoplanin immunohistochemical marker, to identify tumor lymph vessel invasion in cSCC and to assess its expression in tumor cells. METHODS: This was a retrospective case-control study. A series of 101 cSCC, including 51 cases that developed lymphatic metastatic spread (metastasizing cSCC [MSCC]) and 50 cases that resolved definitely after surgical excision (non-MSCC) were included in the study. Lymph vessel invasion using D2-40 was evaluated on all primary biopsy specimens. The percentage of tumor cells showing D2-40 positivity and intensity scoring were recorded. All the immunohistochemical findings were correlated with the clinicopathological features. RESULTS: Lymph vessel invasion was observed in 8% of non-MSCCs and in 25.5% of MSCCs (P = .031). D2-40 expression was significantly increased, both in intensity (odds ratio 4.42 for intensity ++/+++) and in area (odds ratio 2.29 for area >10%), in MSCC when compared with non-MSCC. Interestingly, almost half (49%) of the MSCC had moderate to intense D2-40 positivity compared with 16% of non-MSCC. D2-40 immunohistochemical expression was increased in tumors with an infiltrative pattern of extension. In the multivariate analysis, histologically poorly differentiated tumors, recurrent lesions, and cSCC showing D2-40 overexpression (in intensity) were significantly associated with lymphatic metastases development (odds ratios 15.67, 14.72, and 6.07, respectively). LIMITATIONS: This was a retrospective study. CONCLUSION: The expression of podoplanin associates with high metastatic risk in cSCC.

Angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma of minor salivary glands.

BACKGROUND: Mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. This tumor is characterized by a great variability in clinical behavior, and little is known about the pathological mechanisms involved in its variance. Angiogenesis is an important step in tumor progression and is believed to be an essential event for metastatic dissemination. METHODS: We aimed to investigate angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma measuring the density of neoformed and lymphatic vessels using CD105 and D2-40 antibodies, respectively, and by immunohistochemical evaluation of VEGF-A and VEGF-C proteins. It was also investigated the expression of D2-40 in neoplastic cells. RESULTS: We studied 26 cases of mucoepidermoid carcinoma, which showed great angiogenic activity measured by neoformed vessel density. However, a low density of lymphatics was observed. VEGF-A, VEGF-C, and D2-40 were commonly detected in mucoepidermoid carcinoma, but only VEGF-A expression correlated with neoformed vessel density. Recurrence and nodal metastasis were associated with low VEGF-A expression and low neoformed vessel density, indicating that impaired angiogenesis could lead to an aggressive phenotype. CONCLUSIONS: Angiogenesis seems important in the modulation of mucoepidermoid carcinoma pathogenesis; however, none of the parameters analyzed could predict tumor behavior.

Papillary thyroid carcinoma involving cervical neck lymph nodes: correlations with lymphangiogenesis and ultrasound features.

Stratification of risk factors for cervical lymph node metastasis (LNM) in thyroid papillary carcinoma is important for providing standards for post-operative adjuvant radio-iodine therapy and for patient prognosis. We investigated pathological factors based on the lymphatic vessel system and radiological features associated with tumor with cervical neck LNM. Among patients who had undergone thyroidectomy confirmed to be papillary thyroid carcinoma, we selected 126 age-sex matched paired patients without cervical LNM (group 1) and with LNM (group 2) to evaluate risk factors. Pathological factors evaluated were size, multiplicity, and extra thyroid extension state, based on the pathological reports using stored data. The lymphatic vessel density (LVD) of each tumor was evaluated by staining for VEGFR-3 and D2-40 and correlated with cervical LNM state. Malignant ultrasound features were evaluated to compare the differences between these two groups. Larger tumor size, multiplicity, extrathyroid extension were more common in group 2 (p<0.05). The median percentage of VEGFR-3 for group 1 was 20 (range 0-30) and D2-40 was 13 (range 7-23) while for group 2, VEGFR-3 was 80 (70-90) and D2-40 was 78 (54-114). LVD measured by intratumoral D2-40 staining was 20.6% and 79.4% for group 1 and group 2, respectively. Intra-tumoral lymphatics measured by D2-40 stain had a strong correlation with cervical LNM (Odds 1.230, CI 1.01.-1.499 p value 0.040). Ultrasound (US) features had no significant differences between the two groups although calcifications tended to be higher in group 2 (84% vs. 76% p=0.264). Lymphatic vessel density and nodule echogenicity were not associated with LNM. Intratumoral lymphangiogenesis was most strongly associated with LNM and thus, could be a useful predictive marker for cervical LNM.

The diagnostic utility of D2-40, calretinin, CK5/6, desmin and MOC-31 in the differentiation of mesothelioma from adenocarcinoma in pleural effusion cytology.

OBJECTIVE: To evaluate the utility of the lymphatic endothelial marker D2-40, along with calretinin, CK5/6, desmin and MOC-31, in differentiating mesothelioma and adenocarcinoma in pleural effusion cytology. STUDY DESIGN: Forty-five pleural effusion cases representing confirmed reactive effusions (13), mesotheliomas (11) and metastatic adenocarcinomas (21) were immunostained with antibodies against D2-40, calretinin, CK5/6, desmin and MOC-31. RESULTS: D2-40 showed membranous staining in 82% of mesotheliomas and 77% of reactive effusions. Calretinin and CK5/6 were positive in 100 and 64% of mesotheliomas, and 92 and 31% of reactive effusions, respectively. All adenocarcinomas showed lack of staining with these markers. Desmin was negative in all malignant cases and positive in 85% of reactive effusions. All adenocarcinomas were positive for MOC-31 and negative for the remaining markers. CONCLUSION: Calretinin was the most sensitive in detecting mesothelial differentiation, followed by D2-40. Although useful, D2-40 necessitated cautious interpretation due to occasional focal/weak positivity, particularly in limited cellularity samples. The muscle marker desmin was useful in differentiating benign from malignant effusions but not in distinguishing mesotheliomas from adenocarcinomas. MOC-31 was both highly sensitive and specific for detecting adenocarcinoma and was useful as part of a panel of stains in differentiating cells of mesothelial origin from adenocarcinoma.

Vallecular cyst in an infant: does your specimen show D2-40 immunoreactivity?

Vallecular cysts are infrequent causes of supraglottic obstruction causing stridor and swallowing difficulty in infants. When detected early in life, the management consists of marsupialization or resection. Supraglottic lymphangiomas of the tongue base and vallecula present with similar symptoms and time of presentation. Endoscopic visualization is traditionally considered to be sufficient in identifying and differentiating these. When a vallecular cyst is visually diagnosed by the surgeon during endoscopy, surgical treatment is provided at the same time. Obtaining a specimen is rarely considered for histopathologic diagnostic verification. However, the natural presentation of a cystic lymphangioma may be indistinguishable from a solitary vallecular cyst by endoscopy alone. This case presentation argues in favor of histopathologic diagnosis in vallecular cysts because the 2 may represent a continuum of disease. A vallecular mass with a single large mucus-filled cyst and adjoining edematous soft tissue extension into the tongue base and piriform sinus diagnosed as lymphangioma through D2-40 immunoreactivity is presented.

Histologic assessment of dermatochalasis: elastolysis and lymphostasis are fundamental and interrelated findings.

OBJECTIVE: To determine the presence, degree, and extent of lymphatic, elastic, and collagen fiber alterations in dermatochalasis (DC) specimens. DESIGN: Case control study of patients with DC compared with age-, gender-, and site-matched controls. PARTICIPANTS: A total of 25 eyelid specimens were studied; 15 of these were blepharoplasty specimens (experimental) and 10 were entropion/ectropion specimens of patients without DC (controls). METHODS: The number and maximal dilation of lymphangiectasia was measured by light microscopy, immunohistochemistry with lymphatic marker D2-40, and elastic tissue content by Verhoeff-van Gieson histochemistry. The number of macrophages was compared between patients with DC and controls in CD68 immunostained specimens. MAIN OUTCOME MEASURES: Lymphatic density, edema, and inflammation. RESULTS: Dermatochalasis eyelid specimens showed increased lymphangiectasia density (5.6 vs. 2.4 lymphatics/high power field; P<0.05), maximal lymphatic dilation (127 vs. 51.5 µm; P<0.05), loss of elastic fibers (2.2 vs. 8.9 fibers/high power field; P<0.05), and greater disruption of collagen networks and edema compared with controls (increased stromal collagen bed of 752 vs. 269 µm; P<0.05; increased intercollagen space of 32.5 vs. 11.8 µm; P<0.05). Macrophages were present in greater quantities in DC specimens (28.6 vs. 11.9 macrophages/high power field; P<0.05). CONCLUSIONS: Patients with DC show an increase in number and maximal dilation of lymphatic vessels in conjunction with widely spaced collagen bundles. This finding coexists with loss of elastic fibers, components known to be essential to the structure and function of the lymphatic system. Governed by macrophages, the pathogenesis of DC may begin with subclinical inflammation leading to elastolysis and secondary lymphostasis. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Method for determining the absolute number concentration of nanoparticles from electrospray sources.

We have developed a simple, fast, and accurate method to measure the absolute number concentration of nanoparticles in solution. The method combines electrospray differential mobility analysis (ES-DMA) with a statistical analysis of droplet-induced oligomer formation. A key feature of the method is that it allows determination of the absolute number concentration of particles by knowing only the droplet size generated from a particular ES source, thereby eliminating the need for sample-specific calibration standards or detailed analysis of transport losses. The approach was validated by comparing the total number concentration of monodispersed Au nanoparticles determined by ES-DMA with UV/vis measurements. We also show that this approach is valid for protein molecules by quantifying the absolute number concentration of Rituxan monoclonal antibody in solution. The methodology is applicable for quantification of any electrospray process coupled to an analytical tool that can distinguish monomers from higher order oligomers. The only requirement is that the droplet size distribution be evaluated. For users only interested in implementation of the theory, we provide a section that summarizes the relevant formulas. This method eliminates the need for sample-specific calibration standards or detailed analysis of transport losses.

Expression of IGF-1R, VEGF-C and D2-40 and their correlation with lymph node metastasis in endometrial adenocarcinoma.

Insulin-like growth factor-1 receptor (IGF-1R) regulates carcinogenesis and tumor development and is expressed in normal endometrium. Vascular endothelial growth factor C (VEGF-C) promotes lymph node metastasis. We investigated IGF-1R, VEGF-C and D2-40 in endometrial adenocarcinoma and the association between IGF-1R and lymphatic metastasis, using an immunohistochemical S-P method with 40 cases of endometrial adenocarcinoma and 14 of normal endometrium. IGF-1R expression differed significantly between normal endometrium and adenocarcinoma; it was associated with histological grade but not surgical stage. IGF-1R overexpression was associated with metastasis, but expression was not. VEGF-C expression was greater in normal endometrium than in adenocarcinoma and was associated with metastasis but not with surgical stage or histological grade. IGF-IR and VEGF-C expression were correlated in endometrial adenocarcinoma, and lymphatic vessel density was closely related to both. Abnormal IGF-IR and VEGF-C expression may be important in lymph node metastasis of endometrial adenocarcinoma and might be used to evaluate the prognosis.

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.