Blair Walker

The use of flow cytometry to measure neutrophil function.

Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species (ROS) production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo.

Diagnostic utility of galectin-3 in thyroid cancer.

Galectin-3 (Gal-3), which has received significant recent attention for its utility as a diagnostic marker for thyroid cancer, represents the most well-studied molecular candidate for thyroid cancer diagnosis. Gal-3 is a protein that binds to beta-galactosidase residues on cell surface glycoproteins and has also been identified in the cytoplasmic and nuclear compartment. This marker has been implicated in regulation of normal cellular proliferation and apoptosis, as well as malignant transformation and the metastasis of cancer cells. We here present a mechanistic review of Gal-3 and its role in cancer development and progression. Gal-3 expression studies in thyroid tissue and cytologic tumor specimens and their methodological considerations are also discussed in this article. Despite great variance in their methodology, the majority of immunohistochemical studies found that Gal-3 was differentially expressed in thyroid carcinoma compared with benign and normal thyroid specimens, suggesting that Gal-3 is a good diagnostic marker for thyroid cancer. Recent studies have also demonstrated improved methodological reliability. On the other hand, Gal-3 genomic expression studies have shown inconsistent results for diagnostic utility and are not recommended. Overall, the development of Gal-3 as a diagnostic marker for thyroid cancer represents a promising avenue for future study, and its clinical application could significantly reduce the number of diagnostic thyroid operations performed for cases of indeterminant fine needle aspiration biopsy cytology, and thus positively impact the current management of thyroid nodular disease.

Adenosine A3 receptor stimulation inhibits migration of human eosinophils.

Activation of adenosine A3 receptors (A3‐R) produced a dose‐dependent reduction in the chemotaxis of human eosinophils to platelet‐activating factor (PAF), RANTES, and leukotriene B4 (LTB4) to a maximum of 58,48, and 52%, respectively (P < 0.02). This effect was completely reversed by selective A3‐R antagonists. In contrast, activation of A1 or A2a‐R did not affect PAF‐induced eosinophil chemotaxis. PAF upregulated the expression of CD11b/CD18, down‐regulated L‐selectin, and also increased F‐actin assembly in eosinophils. The expression of these activation markers was not influenced by A3‐R, A2a, or A1‐R stimulation. Activation of A3‐R may play an important role in inflammation by inhibiting eosinophil migration. J. Leukoc. Biol. 62: 465–468; 1997.

Priming and Signal Transduction in Neutrophils

The priming response is a common feature for almost all agents which activate neutrophils. The priming response is associated with a number of events such as degranulation, receptor upregulation, increases in [Ca2+]i and the production of lipid second messengers. For each of these associated events there are data to suggest that priming can also occur in their absence. These contradictions suggest that either the true mechanism for priming has not been found or that there are multiple pathways by which the primed state can be reached. The latter is more likely to be the case.

Complete genomic landscape of a recurring sporadic parathyroid carcinoma

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long‐term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto‐oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto‐oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high‐throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well‐characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient‐matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single‐base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets. Copyright

Lymphoepithelial Cysts of the Pancreas: The Use of Endoscopic Ultrasound-Guided Fine-Needle Aspiration in Diagnosis

Lymphoepithelial cysts (LECs) are rare non-neoplastic lesions that can appear as a complex cyst or a mass in the pancreas. Cytology from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) can be helpful in making a diagnosis with the aim of avoiding unnecessary surgical resection. A case involving a 51-year-old woman with lower abdominal pain who was found to have a multiloculated cystic lesion at the junction of the pancreatic body and tail is described. Cytology from EUS-FNA was consistent with a pancreatic LEC. The lesion was managed conservatively and follow-up imaging of the cyst over the following two years was unchanged. The patient remains clinically well. Cytology from EUS-FNA can help distinguish LECs from cystic neoplasms, thus preventing radical surgical resection of this benign pancreatic cyst.

Hemithyroidectomy is the preferred initial operative approach for an indeterminate fine needle aspiration biopsy diagnosis

BACKGROUND Fine needle aspiration biopsy represents the critical initial diagnostic test used for evaluation of thyroid nodules. Our objectives were to determine the cytological distribution, the utility of clinicopathologic characteristics for predicting malignancy and the true proportion of cancer among individuals who presented with indeterminate cytology and had undergone thyroid surgery for suspicion of cancer. METHODS We retrospectively reviewed 1040 consecutive primary thyroid operations carried out over an 8-year period at a tertiary care endocrine referral centre. Follicular neoplasm (FN), Hürthle cell neoplasm (HN), neoplasms suspicious for but not diagnostic of papillary carcinoma (IP) and neoplasms with cellular atypia (IA) were reviewed. RESULTS In all, 380 individuals presented with cytologically indeterminate thyroid nodules. Of these, 252 (66%) patients had FN, 47 (12%) HN, 44 (12%) IP, 26 (7%) IA and 11 (4%) had mixed diagnoses. Biopsied lesions were found to be malignant on pathological evaluation in 102 (27%) patients: 49 (19%) with FN, 11 (23%) HN, 28 (64%) IP and 9 (35%) with IA. Hemithyroidectomy was adequate definitive treatment in 196 of 225 (87%) patients with FN and 39 of 42 (93%) with HN. Significant associations with a cancer diagnosis were identified for smaller tumour size in patients with FN (p = 0.004) and right thyroid lobe location in patients with IP (p = 0.012), although these factors were nonsignificant in the corrected analyses for multiple comparisons. CONCLUSION In a review of the experience at a Canadian centre, 4 operations were carried out to identify each cancer, and hemithyroidectomy was the optimal initial and definitive surgical approach for most patients.

Preoperative diagnosis of thyroid nodules using the Bethesda System for Reporting Thyroid Cytopathology: a comprehensive review and

Fine-needle aspiration biopsy (FNAB) is the test of choice for the evaluation of nodules, arriving at a cancer diagnosis, and guiding surgical management. This review and meta-analysis aims to objectively evaluate the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) based upon literature reports of histopathological outcomes following cytopathological diagnoses. Thirteen studies were reviewed and the risk of malignancy (ROM) for each of the BSRTC diagnostic categories were calculated as: Non-diagnostic 11–26%, Benign 4–9%, AUS/FLUS 19–38%, FN/SFN 27–40%, SFM 50–79%, and Malignant 98–100%. In typical clinical utilization, the sensitivity and specificity of thyroid FNAB diagnosis using the BSRTC were 96% and 46%, respectively. The BSRTC represents an important advance in standardizing thyroid FNAB cytopathological reporting. Close attention should be paid to the observation that the AUS-FLUS and FN-SFN DCs have overlapping ROMs, and the potential clinical implications of this finding on patient management.

EFFECTS OF ADENOSINE ON GUINEA PIG PULMONARY EOSINOPHILS

Extracellular adenosine has pharmacological activity on a wide variety of cell types and may play an important role as an inflammatory modulator with both pro- and anti-inflammatory activities. These studies examine the effects of adenosine on guinea pig pulmonary eosinophils. Adenosine alone did not directly induce superoxide (O2−) production. Pretreatment with adenosine primed the O2− response of guinea pig pulmonary eosinophils following the addition of 1 or 10μM plateletactivating factor (PAF). Priming was seen at adenosine concentrations greater than 1 μM and was maximal at 100μM. At this maximal dose, adenosine priming increased the O2− response to 1μM and 10μM PAF by 86% and 51%, respectively. Priming by adenosine was not seen when ionomycin or phorbol myristate acid (PMA) were used as agonists. In fura-2 loaded eosinophils, the addition of 100 μM adenosine resulted in a small but significant rise in intracellular calcium of 54.4 ±9.2 nM above baseline. In contrast, similar adenosine concentrations had no effect on cytosolic calcium levels in guinea pig neutrophils. These data demonstrate a pro-inflammatory role for adenosine in elicited guinea pig pulmonary eosinophils.

The genomic and transcriptomic landscape of anaplastic thyroid cancer: implications for therapy

BackgroundAnaplastic thyroid carcinoma is the most undifferentiated form of thyroid cancer and one of the deadliest of all adult solid malignancies. Here we report the first genomic and transcriptomic profile of anaplastic thyroid cancer including those of several unique cell lines and outline novel potential drivers of malignancy and targets of therapy.MethodsWe describe whole genomic and transcriptomic profiles of 1 primary anaplastic thyroid tumor and 3 authenticated cell lines. Those profiles augmented by the transcriptomes of 4 additional and unique cell lines were compared to 58 pairs of papillary thyroid carcinoma and matched normal tissue transcriptomes from The Cancer Genome Atlas study.ResultsThe most prevalent mutations were those of TP53 and BRAF; repeated alterations of the epigenetic machinery such as frame-shift deletions of HDAC10 and EP300, loss of SMARCA2 and fusions of MECP2, BCL11A and SS18 were observed. Sequence data displayed aneuploidy and large regions of copy loss and gain in all genomes. Common regions of gain were however evident encompassing chromosomes 5p and 20q. We found novel anaplastic gene fusions including MKRN1-BRAF, FGFR2-OGDH and SS18-SLC5A11, all expressed in-frame fusions involving a known proto-oncogene. Comparison of the anaplastic thyroid cancer expression datasets with the papillary thyroid cancer and normal thyroid tissue transcriptomes suggested several known drug targets such as FGFRs, VEGFRs, KIT and RET to have lower expression levels in anaplastic specimens compared with both papillary thyroid cancers and normal tissues, confirming the observed lack of response to therapies targeting these pathways. Further integrative data analysis identified the mTOR signaling pathway as a potential therapeutic target in this disease.ConclusionsAnaplastic thyroid carcinoma possessed heterogeneous and unique profiles revealing the significance of detailed molecular profiling of individual tumors and the treatment of each as a unique entity; the cell line sequence data promises to facilitate the more accurate and intentional drug screening studies for anaplastic thyroid cancer.