Microsatellite Instability, Mismatch Repair, and Tumor Mutation Burden in Lung Cancer.

Since US Food and Drug Administration approval of programmed death ligand 1 (PD-L1) as the first companion diagnostic for immune checkpoint inhibitors (ICIs) in non-small cell lung cancer, many patients have experienced increased overall survival. To improve selection of ICI responders versus nonresponders, microsatellite instability/mismatch repair deficiency (MSI/MMR) and tumor mutation burden (TMB) came into play. Clinical data show PD-L1, MSI/MMR, and TMB are independent predictive immunotherapy biomarkers. Harmonization of testing methodologies, optimization of assay design, and results analysis are ongoing. Future algorithms to determine immunotherapy eligibility might involve complementary use of current and novel biomarkers. Artificial intelligence could facilitate algorithm implementation to convert complex genetic data into recommendations for specific ICIs.

Haemolysis index conundrum in a COVID-19 patient.

In this study, we report a preanalytical challenge noted in our laboratory on plasma samples from a critically ill COVID-19 patient treated with hydroxychloroquine. This is significant because, in critically ill COVID-19 patients on hydroxychloroquine, plasma samples can have a high measured haemolysis index in the absence of haemolysis, with the impact on reporting the results for potassium and other analytes.

Plasma cell leukemia with soft tissue involvement; reporting a rare case.

Plasma cell leukemia (PCL) is a rare aggressive variant of multiple myeloma. PCL is diagnosed when clonal plasma cells constitute more than 20 % of the total circulating leukocytes or when the absolute plasma cell count exceeds 2 × 109 /L. Extramedullary involvement including cavity effusion is frequently seen at the time of diagnosis. However, soft tissue involvement is rarely encountered with only one published case in the English literature. We report a 74-year-old man, who presented with progressive shortness of breath over a few months. Laboratory studies showed leukocytosis (32 × 109 /L) with 26 % peripheral plasmacytoid cells and significantly elevated lactate dehydrogenase (> 2500 U/L). Serum protein electrophoresis detected a monoclonal IgG lambda band. A 7.4 cm left hilar mass, bilateral pleural effusion, and multiple fluorodeoxyglucose (FDG)-avid subcutaneous nodules in the pelvic and gluteal regions were demonstrated on imaging. Gluteal nodule biopsy revealed diffuse infiltrative CD138+ and MUM1+ cells with aberrant CD4, CD30, and BCL2 expression. The Ki-67 proliferation index was 70 %. Bone marrow biopsy showed sheets of atypical plasma cells with lambda-restriction and CD138 and MUM1 expression without cyclin D1 and CD20 expression. These cells comprise approximately 70-80 % of the bone marrow cellularity. A similar immunophenotype was demonstrated in peripheral and bone marrow flow cytometry. Molecular and cytogenetics showed an abnormal clone with a complex karyotype including monosomy 13 and 14q deletion. Overall, these findings are consistent with a plasma cell neoplasm. Our case study illustrates soft tissue involvement in PCL, which is rarely seen.

Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein.

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.

Abnormalities of uterine cervix in women with inflammatory bowel disease.

AIM: To evaluate the prevalence of abnormalities of the uterine cervix in women with inflammatory bowel disease (IBD) when compared to healthy controls. METHODS: One hundred and sixteen patients with IBD [64 with Crohn's disease (CD) and 52 with ulcerative colitis (UC)] were matched to 116 healthy controls by age (+/- 2 years) at the time of most recent papanicolaou (Pap) smear. Data collected consisted of age, race, marital status, number of pregnancies, abortions/miscarriages, duration and severity of IBD, Pap smear results within five years of enrollment, and treatment with immunosuppressive drugs. Pap smear results were categorized as normal or abnormal including atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LGSIL), and high-grade squamous intraepithelial lesion (HGSIL). RESULTS: The median age at the time of Pap smear was 46 (range: 17-74) years for the IBD group and matched controls (range: 19-72 years). There were more Caucasian subjects than other ethnicities in the IBD patient group (P = 0.025), as well as fewer abortions (P = 0.008), but there was no significant difference regarding marital status. Eighteen percent of IBD patients had abnormal Pap smears compared to 5% of controls (P = 0.004). Subgroup analysis of the IBD patients revealed no significant differences between CD and UC patients in age, ethnicity, marital status, number of abortions, disease severity, family history of IBD, or disease duration. No significant difference was observed in the number of abnormal Pap smears or the use of immunosuppressive medications between CD and UC patients (P = 0.793). No definitive observation could be made regarding HPV status, as this was not routinely investigated during the timeframe of our study. CONCLUSION: Diagnosis of IBD in women is related to an increased risk of abnormal Pap smear, while type of IBD and exposure to immunosuppressive medications are not. This has significant implications for women with IBD in that Pap smear screening protocols should be conscientiously followed, with appropriate investigation of abnormal results.

Local shear conditions and platelet aggregates regulate the incorporation and activity of circulating tissue factor in ex-vivo thrombi.

The presence of thrombogenic blood-borne or circulating tissue factor (cTF) has recently been demonstrated. These observations have implicated cTF to be a key determinant of thrombus propagation by depositing on platelets in nascent thrombi. Previously, we detected cTF by detergent solubilization and addition of phospholipids. We now report the direct demonstration of TF activity in ex-vivo thrombi. Collagen-coated substrates were exposed to native blood at shear rates of 0, 650, and 2,000 s(-1) for 10 min in a modified rotating Teflon cone and plate viscometer. Substrates were then gently rinsed to remove 'loose' (unadherent) components of blood. cTF activity was measured by adding a solution containing 10 nM FVIIa, 100 nM FX, and 5 mM CaCl(2) to the substrates exposed to blood. Samples of this mixture were obtained at intervals for 30 min and the amount of Xa generated was quantified by adding a chromogenic substrate, Spectrozyme Xa, and measuring the increase in OD at 405 nm. Our studies show that a minimal amount of generated Xa (approximately 1nM) can be measured from ex-vivo thrombi. Static and shear samples generated the same amount of Xa, with the exception of blood subjected to 650 s(-1) shear. At 650 s(-1) shear rate, the amount of Xa generated reached a maximum of 4 nM at 5 min and then decreased to approximately 1 nM. Immunohistological stains and fluorescent images demonstrate the presence of cTF antigen at 650 s(-1) wall shear rate.

Molecular testing in lung cancer: fine-needle aspiration specimen adequacy and test prioritization prior to the CAP/IASLC/AMP Molecular Testing Guideline publication.

BACKGROUND: Subtyping of lung carcinoma with immunohistochemistry is essential for diagnosis, whereas molecular testing (MT) is required for therapy guidance. In the current study, the authors report on MT performed on fine-needle aspiration specimens at the study institution over a 2-year period preceding the April 2013 College of American Pathologists (CAP)/International Association for the Study of Lung Cancer (IASLC)/Association for Molecular Pathology (AMP) Molecular Testing Guideline (MTG) publication. METHODS: The database of the study institution was retrospectively queried for cases of lung and thoracic/lower cervical lymph node fine-needle aspiration specimens for 2011 through 2012. RESULTS: Of 246 selected cases, 26 featured a limited amount of material in cell blocks. MT increased significantly between 2011 and 2012 and was requested in 39.4% of cases (97 of 246 cases): 86 of those cases had at least 1 MT result and 11 had insufficient material for any MT. Anaplastic lymphoma kinase (ALK) testing was performed in 9 cases in which DNA was insufficient for epidermal growth factor receptor (EGFR) testing. In addition, 13 cases of adenocarcinoma/non-small cell lung carcinoma had at least 1 MT canceled because of insufficient DNA, but at the same time had an average of 3.46 immunohistochemical stains performed. CONCLUSIONS: Of all the cytology specimens, 10.6% featured limited material; however, no universally accepted testing sequence priority was available at the time the study was performed. As per the MTG, MT should take precedence over immunohistochemistry in cases of adenocarcinoma/non-small cell lung carcinoma. Approximately 5.3% of the specimens in the current study had insufficient material for MT while having multiple stains performed instead. The MTG also recommend performing EGFR before ALK testing; the authors found 9 cases with insufficient material for EGFR testing that had ALK testing performed. The results of the current study underscore the need for a testing prioritization algorithm in view of the MTG publication to serve as reference for both clinicians and pathologists.

Inherited hearing loss: molecular genetics and diagnostic testing.

BACKGROUND: Hearing loss is a clinically and genetically heterogeneous condition with major medical and social consequences. It affects up to 8% of the general population. OBJECTIVE: This review recapitulates the principles of auditory physiology and the molecular basis of hearing loss, outlines the main types of non-syndromic and syndromic deafness by mode of inheritance, and provides an overview of current clinically available genetic testing. METHODS: This paper reviews the literature on auditory physiology and on genes, associated with hearing loss, for which genetic testing is presently offered. RESULTS/CONCLUSION: The advent of molecular diagnostic assays for hereditary hearing loss permits earlier detection of the underlying causes, facilitates appropriate interventions, and is expected to generate the data necessary for more specific genotype-phenotype correlations.

Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.