Development and validation of a simple method for the extraction and quantification of crisaborole in skin layers.

Crisaborole is a boron compound recently approved by the US Food and Drug Administration as a 2% ointment for the treatment of mild to moderate atopic dermatitis. This work describes a simple method for the quantification of the drug in the skin layers at the end of in-vitro permeation experiments. Chromatographic separation was carried out on a reverse-phase C18 column using a mixture of trifluoroacetic acid 0.05%-acetonitrile (55:45, v/v) as mobile phase, pumped at 1 ml/min. Column temperature was 35°C and UV detection was performed at 250 nm. The method was linear in the range of concentration from 0.06 to 6 µg/ml (R2 = 1) and was selective, precise and accurate. Depending on the solvent used, the LOQ ranged from 0.014 to 0.030 µg/ml and the LOD from 0.005 to 0.010 µg/ml. The extraction from all the skin layers was quantitative. The developed method was successfully tested in an in-vitro permeation study, proving to be an effective tool in the development of new formulations containing crisaborole.

Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature.

AIM: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). METHODS AND RESULTS: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. CONCLUSIONS: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.

An Alternative Device for the Topical Treatment of Oral Cancer: Development and Ex-Vivo Evaluation of Imiquimod-Loaded Polysaccharides Formulations.

The topical use of imiquimod (IMQ), a non-specific immune response modifier, showed to be a promising therapeutic option for the early-stage treatment of some type of oral cancer, even when performed with a formulation (Aldara®) developed and approved for skin application. The aim of this work was the development of buccal formulations for the topical administration of IMQ with improved mucosal retention and reduced trans-mucosal permeation when compared to the reference formulation. Three different hydrogels based on carboxymethyl chitosan (CMChit), sodium alginate (A), and xanthan gum (X) in different combinations were prepared, and the loading of imiquimod was successfully performed by using a micellar formulation based on d-α-tocopheril polyethylene glycol 100 succinate (TPGS). Except for CMChit formulation, in all the other cases, the performance in vitro on the mucosa resulted comparable to the commercial formulation, despite the drug loading being 50-fold lower. Converting the gels in films did not modify the IMQ accumulated with respect to the correspondent gel formulation but produced as a positive effect a significant reduction in the amount permeated. Compared to the commercial formulation, this reduction was significant (p < 0.01) in the case of X film, resulting in an improvement of the retained/permeated ratio from 1 to 5.44. Mucoadhesion evaluation showed similar behavior when comparing the developed gels and the commercial formulation, and an excellent bioadhesion was observed for the films.

Validation of a HPLC-UV method for the quantification of budesonide in skin layers.

A simple and sensitive HPLC method for the quantification of budesonide in skin layers was developed and validated. Budesonide was extracted from stratum corneum, epidermis and dermis by means of a mixture of acetonitrile:water (recovery > 90%). Budesonide quantification was performed with a RP-C18 column using methanol and water mixture (69:31, v/v) as mobile phase, pumped at 0.8 ml/min. The absorbance was monitored at 254 nm. The method resulted to be selective, linear in the range 0.05-5 or 10 µg/ml, precise and accurate. LLOQ resulted to be 0.05 µg/ml. The developed method appeared to be appropriate for the quantification of budesonide in skin layers at the end of in vitro permeation experiments since the recovery of the applied dose was 97 ± 1%, in line with requirement of the OECD guideline for the testing of the chemicals (Skin absorption: in vitro method).

In Vitro Skin Retention of Crisaborole after Topical Application.

Crisaborole, a nonsteroidal phosphodiesterase 4 inhibitor, represents the first nonsteroidal medication approved for the treatment of atopic dermatitis in over a decade. In this work, crisaborole skin permeation and retention was studied in vitro from a 2% ointment using porcine skin as barrier. Crisaborole was also characterized in terms of thermal behavior, solubility, and logP. Control experiments were performed also on tape stripped skin to clarify the role of stratum corneum in drug partitioning and permeation across the skin. The results obtained indicate that crisaborole accumulates into the skin in considerable amounts after application of a topical lipophilic ointment. Crisaborole shows more affinity for the dermis compared to the epidermis despite its relatively high value of partition coefficient; stratum corneum analysis revealed a low affinity of the drug for this skin layer. Skin penetration across hair follicles or sebaceous glands can be a reason for the high dermis retention and is worth further investigation. The comparison with data obtained from a solution in acetonitrile suggests that the formulation plays a certain role in determining the relative distribution of crisaborole in the skin layers and in the receptor compartment.

Flow cytometry identification of nonhemopoietic neoplasms during routine immunophenotyping.

INTRODUCTION: Nonhemopoietic neoplasms (NHNs) may be encountered during routine flow cytometry (FC) immunophenotyping. The clue of their presence mainly relies on detection of CD45-negative (CD45-) cells with altered scatter parameters. METHODS: In this study, we evaluated a monoclonal antibody combination conceived to characterize the CD45- population by FC, suspected of belonging to NHNs, when present. The panel included CD45 for leucocytes identification, CD326 (clones BerEP4 and HEA-125) to mark epithelial cells, CD33 to identify myeloid cells, CD138 to trace plasma cells and CD56 useful in the identification of neuroendocrine tumours. 7AAD vital dye was used to gate out dead cells. Results were correlated with cytomorphology and confirmed by histological data, if available. RESULTS: Among 9422 specimens submitted for routine FC investigation, 47 samples that included fine-needle aspirates, bone marrow aspirates, tissue biopsies and body fluids had a detectable CD45- population and a sufficient cell amount to be further investigated. FC revealed the presence of CD326-positive epithelial cells in 38 specimens; altered scatter parameters and variable reactivity to the other antigens tested allowed to suspect NHNs in the remaining nine samples. The presence of NHNs was confirmed in all cases by morphology. CONCLUSIONS: The current results show that when CD45- cells with altered scatter parameters were detected, cytometrists involved in leukaemia/lymphoma diagnosis may require further FC investigations to rapidly identify NHNs in different specimens, thus reducing the time of the immunohistochemical diagnostic workup to reach a final diagnosis.

The usefulness of flow cytometric CD10 detection in the differential diagnosis of peripheral T-cell lymphomas.

We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.

Post-remissional and pre-transplant role of minimal residual disease detected by WT1 in acute myeloid leukemia: A retrospective cohort study.

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) is still under investigation. The aim of the present retrospective study was to assess the role of Wilms tumor gene 1 (WT1) overexpression in a large monocentric cohort of AML patients. Among 255 enrolled patients, MRD was investigated in those in complete remission (CR) with an available WT1 at baseline (>250 copies) and at two further time-points: after induction (n=117) and prior allogeneic hematopoietic cell transplantation (allo-HCT), n=65. Baseline BM WT1 overexpression was not associated with response to induction (p=0.244). Median overall survival (OS) and disease-free survival (DFS) were significantly shorter in patients with >350 WT1 copies after induction compared to those with ≤350 (HR for mortality 2.13; 95% CI 1.14-3.97, p=0.018 and HR for relapse 2.81; 95% CI 1.14-6.93, p=0.025). Patients with WT1>150 copies pre allo-HCT had a significantly higher 2-year cumulative incidence of relapse (CIR) compared to those with WT1≤150 (HR 4.61; 95% CI 1.72-12.31, p=0.002). The prognostic role of WT1 overexpression resulted independent from other well-established risk factors. According to these results, WT1 overexpression might represent an additional MRD tool for risk stratification in patients classified nowadays in CR.

Effect of high-risk human papillomavirus oncoproteins on p53R2 gene expression after DNA damage.

The p53R2 protein is a p53-inducible small subunit of ribonucleotide reductase. It plays a crucial role in p53-dependent cellular response to DNA damage and oxidative stress by providing deoxyribonucleotides (dNTPs) to the DNA repair machinery and by scavenging reactive oxygen species (ROS). To investigate the effects of high-risk human papillomavirus (HPV) oncoproteins on p53R2 expression after DNA damage, we analyzed the p53R2 protein levels in human cells ectopically expressing the HPV-16 E6 and E7 genes, and in the HPV-positive cancer cell lines SiHa, CaSki and HeLa, exposed to adriamycin or to H(2)O(2). We found that in normal cells, p53R2 expression is efficiently induced by both H(2)O(2) and adriamycin, supporting the role of p53R2 in cellular response to oxidative stress. Ectopic expression of E6 impaired p53 and p53R2 induction after DNA damage in human fibroblasts. Moreover, SiHa, CaSki and HeLa cells were unresponsive to H(2)O(2) exposure, and adriamycin induced p53R2 levels only in SiHa cells. Our results imply that high-risk HPV infection may suppress the p53R2-dependent dNTPs supply to the DNA repair system and the ROS scavenging activity; they also suggest that an altered p53R2 response to genotoxins and to oxidative stress may contribute to HPV-induced genetic instability and carcinogenesis.

Extranodal Lymphoproliferative Processes and Flow Cytometry.

OBJECTIVE: Fine-needle aspiration (FNA) cytology is a safe and cost-effective technique for the diagnosis of lymphoproliferative processes, especially when correlated with clinical and imaging studies. However, cytology alone may be unable to detect a lymphoid neoplastic process, as architectural features are less obvious than in histologic preparations and, in certain cases, reactive processes may mimic lymphoma. Flow cytometry (FC) has been recognized as an important ancillary technique in the diagnosis of lymphoid neoplasms and it can be used in conjunction with FNA in the evaluation of lymphoproliferative processes. STUDY DESIGN: We performed a review of the published literature concerning FC applied to the detection of salivary glands and thyroid lymphoproliferative processes, which are frequently related to autoimmune diseases and difficult to diagnose by cytomorphology alone. RESULTS: FC is able to detect and subtype non-Hodgkin lymphomas and may contribute to the exclusion of a neoplastic process in cytologically unclear cases. CONCLUSIONS: FC can be successfully applied in the differential diagnosis of lymphoproliferative processes in the head and neck region. The FNA-FC combined approach can reduce time to therapy and may prevent unnecessary surgical biopsies.

Single-Tube Flow Cytometry Assay for the Detection of Mature Lymphoid Neoplasms in Paucicellular Samples.

OBJECTIVES: Flow cytometry (FC) has become a useful support for cytomorphologic evaluation (CM) of fine-needle aspirates (FNA) and serous cavity effusions (SCE) in cases of suspected non-Hodgkin lymphoma (NHL). FC results may be hampered by the scarce viability and low cellularity of the specimens. STUDY DESIGN: We developed a single-tube FC assay (STA) that included 10 antibodies cocktailed in 8-color labeling, a cell viability dye, and a logical gating strategy to detect NHL in hypocellular samples. The results were correlated with CM and confirmed by histologic or molecular data when available. RESULTS: Using the STA, we detected B-type NHL in 31 out of 103 hypocellular samples (81 FNA and 22 SCE). Of these, 8 were not confirmed by CM and 2 were considered to be only suspicious. The FC-negative samples had a final diagnosis of benign/reactive process (42/72), carcinoma (27/72), or Hodgkin lymphoma (3/72). CONCLUSIONS: The STA approach allowed obtainment of maximum immunophenotyping data in specimens containing a low number of cells and a large amount of debris. The information obtained by STA can help cytomorphologists not only to recognize but also to exclude malignant lymphomas.

Utilility of flow cytometry as ancillary study to improve the cytologic diagnosis of thyroid lymphomas.

BACKGROUND: To evaluate the efficacy of the use of flow cytometry (FC) immunophenotyping together with fine-needle aspiration cytology (FNAC) in the diagnosis of thyroid lymphoma. METHODS: FC was performed in parallel with FNAC in 35 samples of suspected thyroid lymphoma over a 12 years period. Results were correlated with histological or molecular findings and follow-up, when available. RESULTS: A final diagnosis of lymphoma was given in 13 of 35 (37.1%) specimens. Among the 22 cases considered negative for lymphoma by FC, 11 were diagnosed as thyroiditis by cytology, 7 as reactive, 2 were anaplastic carcinoma, and 2 cases were considered cytologically suspicious for lymphoma but were not confirmed by further investigations. Histology on core biopsy or molecular analysis was available in 12 of 13 lymphoma cases (92.3%). Data obtained by the combination cytology/FC were confirmed in all cases on histology biopsies. Correlation with histology showed a sensitivity and a specificity of 100% for the combination cytology/FC. CONCLUSIONS: FC is an important additional test that can contribute with cytology to the identification of lymphomas of the thyroid. FC can detect the presence of small neoplastic lymphocyte populations and may contribute to the diagnosis of cases in which the lymphoid infiltrate is difficult to interpret on cytology alone.

Immunophenotyping of paucicellular samples.

Immunophenotyping of paucicellular samples may represent a diagnostic challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the number of tests that can be performed. Specimens such as fine needle aspirates (FNA), human body fluids (BF), cerebrospinal fluid (CSF), or ocular fluid (OF) sent for FC investigations in the case of suspicion of lymphoma, or for lymphoma monitoring, may contain very low numbers of cells. In these cases, it is mandatory to obtain the largest amount possible of useful information from a single tube. The basic protocol described in this unit provides a method that combines the use of multiple monoclonal antibodies (MAbs) with a Boolean gating strategy to identify and quantify the main lymphocyte populations, as well as to detect lymphomatous B cells or any aberrant T cell expression, if present, in paucicellular samples.

Tissue flow cytometry immunophenotyping in the diagnosis and classification of non-Hodgkin's lymphomas: a retrospective evaluation of 1,792 cases.

A retrospective analysis of 1,792 solid tissues suggestive of lymphoma, submitted over a 12-year period, was carried out and flow cytometry (FC) results were compared with histologic findings. The final histologic diagnosis of cases documented in this report is as follows: 1,270 non-Hodgkin's lymphomas (NHL); 17 composite lymphomas; four NHL plus carcinomas; five post-transplant lymphoproliferative disorders; 105 Hodgkin's lymphomas (HL); eight acute leukemias; 42 tissue cancers; and 341 non-neoplastic diseases. A strong correlation between morphology and FC data was observed among hematological malignancies (1,268/1,304, 97.2%) with the exception of HL. Among B-NHL, FC detection of clonally restricted B-cell allowed the identification of lymphomas that were not histologically clear and the differential diagnosis between follicular lymphoma and reactive hyperplasia. A high correlation level (r = 0.83; P < 0.0001) was obtained in comparing proliferation results obtained by FC and immunohistochemistry. Among T-NHL, FC detection of an aberrant phenotype direct histologic diagnosis in cases having less than 20% of neoplastic cells. In nine cases, FC suggested the need to evaluate a neoplastic population, not morphologically evident. Results show that FC routinely performed on tissue samples suspected of lymphomas is a fundamental adjunct to morphology in the diagnosis of NHL and may enhance the performance of the histologic evaluation so as to achieve the final diagnosis. To the best of our knowledge, this is the first report in the literature of a wide series of tissues also studied by FC.

Flow cytometric detection of liposomal cytarabine in cerebrospinal fluid of patients treated with intrathecal chemotherapy.

We report the unusual flow cytometric detection of liposomes in the cerebrospinal fluid of patients with aggressive non-Hodgkin's lymphoma and acute myeloid leukemia, treated with intrathecal liposomal cytarabine.

Ten antibodies, six colors, twelve parameters: a multiparameter flow cytometric approach to evaluate leptomeningeal disease in B-cell non-Hodgkin's lymphomas.

BACKGROUND: Flow cytometry (FC) is considered a sensitive and specific technique for the detection of occult lymphoma cells in cerebrospinal fluid (CSF). METHODS: The diagnostic sensitivity of a FC approach which uses a combination of 10 antibodies, a single-tube evaluation, and a six-color instrument, was evaluated and compared to conventional cytology (CC) for the detection of lymphomatous cells in the CSF of 44 patients affected by B-cell non-Hodgkin's lymphoma (B-NHL) considered at high risk of central nervous system spread. RESULTS: The CSF obtained from 36 newly diagnosed and 8 relapsed patients affected by B-cell lymphoma was assessed by FC and CC on a total of 62 samples; 52/62 (82.6%) were considered paucicellular as they had fewer than 10 cells/µl. All cases were evaluated by both methods. FC gave 15/62 (24%) positive results, CC 10/62 (16%) positive results; none of the samples evaluated had a positive CC with a negative FC result. CONCLUSIONS: The use of a multiparameter FC approach, which collects an elevated number of monoclonal antibodies in a single tube and identify different cell populations with a selective gating strategy analysis, allows for the evaluation of lymphocyte subsets and the detection of leptomeningeal disease in B-NHL, even in the presence of paucicellularity of samples.

Flow cytometric detection of degranulated basophils in chronic myeloid leukemia in accelerated phase.

We report a rare case of chronic myeloid leukemia in accelerated phase with basophilic transformation, in which basophils exceeding 70%, were detectable only by flow cytometry because of their morphologic atypicality and degranulation.

Usefulness of multiparametric flow cytometry in detecting composite lymphoma: study of 17 cases in a 12-year period.

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.