Alterations in Pre/Post Oxygenator Flows Due to Fibrin Deposition in the CardioHelp System-A Case Report.

We present a 62-year-old patient with COVID-19 pneumonia on Veno-venous (VV) Extracorporeal Membrane Oxygenation (ECMO) with unique perturbations to pre and post oxygenator pressures due to fibrin deposition in despite being on a Heparin/Bivalirudin infusion and activated Partial Thromboplastin Time (aPTT) within therapeutic range of 60-80 seconds. On Day 8 of ECMO support, it was noticed that flows steadily decreased despite unchanged RPMs. Unlike typical blood flow to circuit pressure relationships, the circuit pressures did not correlate with the observed decreased flow. The Delta Pressure (ΔP) was not elevated. The patient's vitals were stable. On inspection post change-out, clots were noted in the oxygenator outlets. Oxygenator clots are usually associated with increased ΔP. In this scenario, clots in the oxygenator blocked 1 of the 4 outlets in the oxygenator causing the flow, pressures, and ΔP to drop consecutively. Due to reduced flow, the ΔP was not elevated despite extensive clots. The fibrin clot location in the CardioHelp ECMO circuit may lead to unexpected pressure and flow alterations. Sole reliance on ΔP as a marker for oxygenator clots may be misleading. Careful monitoring and timely diagnosis of coagulation status may lead to changes in anticoagulation goals and meaningfully impact patient outcomes.

DNA aneuploidy and tissue architecture in oral potentially malignant disorders with epithelial dysplasia assessed by a 10 locus FISH panel.

Subjectivity in oral dysplasia grading has prompted evaluation of molecular­based tests to predict malignant transformation. Aneuploidy detected by DNA image­based cytometry (ICM) is currently the best predictor but fails to detect certain high risk lesions. A novel multiplex fluorescence in situ hybridization (FISH) panel was used to explore possible explanations by detecting aneuploidy at the single cell level. FISH was compared to reference standard DNA ICM in 19 oral lesions with epithelial dysplasia and used to characterize the cellular architecture. Copy number variation at 3q28, 7p11.2, 8q24.3, 11q13.3 and 20q13.12 and matched chromosome specific loci were assessed by dual­color FISH to assess numerical and spatial patterns of copy number increase and gene amplification. FISH revealed wide variation in copy number at different loci. Only low level copy number gain was present and often in only a small proportion of cells, although usually with all or all but one locus (9/12). Four cases showed gene amplification, one at two loci. Some probes revealed an internal presumed clonal structure within lesions not apparent in routine histological examination. Both methods produced similar diagnostic results with concordance in detection of aneuploidy by both methods in 17 out of 19 samples (89%). We have shown that oral dysplastic lesions may contain very few aneuploid cells at a cellular level, high copy number gain is rare and changes appear to arise from large chromosomal fragment duplications. Single stem lines are relatively homogeneous for loci with copy number gain but there is a subclonal structure revealed by gene amplification in some lesions.

Prevalence of Fecal Incontinence in the Acute Care Setting.

PURPOSE: The aim of this study was to determine the prevalence of fecal incontinence (FI) and its associated risk factors in acutely ill adult hospitalized patients. METHODS: A cross-sectional design was used to collect data at 2 time points in 7 hospitals in the Midwestern United States. An investigator-developed tool was used by trained data collectors to identify pertinent patient characteristics, the presence of FI, and potential associated factors. RESULTS: The prevalence of FI in the 1083 patients assessed was 20% (n = 221). Prevalence rates from the 7 individual hospitals ranged from 16% to 30%. Medications were the most common associated factor (49%; n = 109), followed by neurologic diseases (40%; n = 89), and bowel motility disorders (30%; n = 67). The majority of patients with FI had stool consistency described as "loose unformed" (59%; n = 130) or "liquid" (25%; n = 55). Many patients had multiple potential risk factors for FI; 48% (n = 107) had 1 associated factor, 37% (n = 82) had 2 associated factors, and 8% (n = 18) had 3 or more associated factors. Age was associated with an increased likelihood of FI; the chances for FI increase 1.7% with each year of age. Unit type was also a significant associated with FI; patients managed in the intensive care unit were 78% more likely to have FI as compared to patients care for in a medical-rehabilitation unit. CONCLUSIONS: Fecal incontinence is a common problem in hospitalized adult patients. Previously identified risk factors were also found in our sample.

Patients with HPV-related tonsil squamous cell carcinoma rarely harbour oncogenic HPV infection at other pharyngeal sites.

OBJECTIVES: Patients with human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) have a reduced risk of developing second primary upper aerodigestive tract (UADT) tumours compared to patients with HPV-negative primary tumours at the same site. To determine whether this finding might be explained by a lack of viral-induced field cancerisation or multifocal infection, we investigated whether there was epithelial dysplasia and/or evidence of HPV infection at other pharyngeal mucosal sites in patients presenting with the disease. MATERIALS AND METHODS: Sixty-three patients with primary tonsil SCC and 108 pharyngeal endoscopic biopsies, representing at least one pharyngeal subsite from each patient, were included in this study. Tissue samples were tested using HPV PCR (GP5+/6+), p16 immunohistochemistry (IHC) and high risk HPV DNA in situ hybridisation (ISH). RESULTS: There were 46 patients with HPV-related SCC and 17 patients with HPV-negative disease. PCR detected HPV DNA in a fifth of pharyngeal endoscopic biopsies and was equally likely to be from a patient with HPV-related SCC as from a patient with HPV negative disease. All PCR positive cases were tested using p16 IHC and high risk HPV ISH and only three biopsies were positive. Significantly, these three biopsies all showed evidence of epithelial dysplasia and were from patients with an HPV positive index tumour. CONCLUSION: Our data suggest that virus-induced field cancerisation and/or multifocal oncogenic HPV infection of the pharynx is uncommon in OPSCC and supports the concept that these patients have a lower risk of developing second primary tumours of the UADT.

CD44 expression in oro-pharyngeal carcinoma tissues and cell lines.

Expression of CD44, a transmembrane hyaluronan-binding glycoprotein, is variably considered to have prognostic significance for different cancers, including oral squamous cell carcinoma. Although unclear at present, tissue-specific expression of particular isoforms of CD44 might underlie the different outcomes in currently available studies. We mined public transcriptomics databases for gene expression data on CD44, and analyzed normal, immortalized and tumour-derived human cell lines for splice variants of CD44 at both the transcript and protein levels. Bioinformatics readouts, from a total of more than 15,000 analyses, implied an increased CD44 expression in head and neck cancer, including increased expression levels relative to many normal and tumor tissue types. Also, meta-analysis of over 260 cell lines and over 4,000 tissue specimens of diverse origins indicated lower CD44 expression levels in cell lines compared to tissue. With minor exceptions, reverse transcribed polymerase chain reaction identified expression of the four main isoforms of CD44 in normal oral keratinocytes, transformed lines termed DT and HaCaT, and a series of paired primary and metastasis-derived cell lines from oral or pharyngeal carcinomas termed HN4/HN12, HN22/HN8 and HN30/HN31. Immunocytochemistry, Western blotting and flow cytometric assessments all confirmed the isoform expression pattern at the protein level. Overall, bioinformatic processing of large numbers of global gene expression analyses demonstrated elevated CD44 expression in head and neck cancer relative to other cancer types, and that the application of standard cell culture protocols might decrease CD44 expression. Additionally, the results show that the many variant CD44 exons are not fundamentally deregulated in a diverse range of cultured normal and transformed keratinocyte lines.

Human papillomavirus detection in dysplastic and malignant oral verrucous lesions.

The role of human papillomaviruses (HPV) in dysplastic and malignant oral verrucous lesions is controversial since there is a wide range in the incidence of virus detection. This study used a multi-tiered method of HPV detection using DNA in-situ hybridisation (ISH) for low- and high-risk subtypes, consensus PCR, and HPV genotype analysis in archival tissue from 20 cases of dysplastic and malignant oral verrucous lesions. The biological significance of HPV DNA detection was assessed by p16 immunohistochemistry (IHC). While 1/7 carcinomas and 5/13 dysplasias contained HPV DNA by consensus PCR and genotype analysis, all specimens were negative for low- and high-risk HPV ISH and negative for p16 IHC. Results show that although high-risk HPV DNA is detectable in a subset of these lesions, the lack of p16 overexpression suggests that the oncogenic process is not driven by HPV oncoproteins.

Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM), and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE). Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH) analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used to increase power and compensate for increased error rates.

Evaluation of human papillomavirus testing for squamous cell carcinoma of the tonsil in clinical practice.

BACKGROUND: Oncogenic human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC) is a subtype of head-and-neck cancer with a distinct clinical and prognostic profile. While there are calls to undertake HPV testing for oropharyngeal SCCs within the diagnostic setting and for clinical trials, there are currently no internationally accepted standards. METHODS: 142 tonsil SCCs were tested using p16 immunohistochemistry (IHC), high-risk HPV DNA in situ hybridisation (ISH) and HPV DNA polymerase chain reaction (PCR; GP5+/6+ primers). RESULTS: There were high levels of agreement between pathologists for p16 IHC and HPV ISH scoring; however, around 10% of HPV ISH cases showed some interobserver discrepancy that was resolved by slide review. The combination of p16 IHC and HPV ISH classified 53% of the samples as HPV-positive, whereas the combination of p16 IHC and HPV PCR classified 61% of the samples as HPV-positive. By employing a three-tiered, staged algorithm (p16 IHC/HPV ISH/HPV PCR), the authors were able to classify 98% of the cases as either HPV-positive (p16 IHC+/HPV DNA+; 62%) or HPV-negative (p16 IHC-/HPV DNA-; 35%). CONCLUSIONS: The current study suggests that using a combination of p16 IHC/HPV ISH/HPV PCR, in a three-tiered, staged algorithm, in conjunction with consensus reporting of HPV ISH, leads to less equivocal molecular classification. In order to ensure consistent reporting of this emerging disease, it is increasingly important for the head-and-neck oncology community to define the minimum requirements for assigning a diagnosis of 'HPV-related' oropharyngeal SCC in order to inform prognosis and for stratification in clinical trials.

Expression profiles and clinical correlations of degradome components in the tumor microenvironment of head and neck squamous cell carcinoma.

PURPOSE: Head and neck squamous cell carcinomas (HNSCC) are characterized by high morbidity and mortality, largely due to the high invasive and metastatic potential of these tumors, high recurrence rates, and low treatment responses. Proteinases have been implicated in several aspects of tumor growth and metastasis in a broad range of tumors including HNSCC. EXPERIMENTAL DESIGN: Comprehensive expression profiling of proteinases [matrix metalloproteinases (MMPs), A disintegrin and metalloproteinase (ADAMs), and ADAMs with thrombospondin motif (ADAMTSs)] and their inhibitors [tissue inhibitor of metalloproteinases (TIMPs)] was done using quantitative real-time reverse transcription-PCR analysis of a large cohort of tissue samples representing the tumor (n = 83), the invasive margin (n = 41), and the adjacent tissue (n = 41) from 83 HNSCC patients, along with normal tissue controls (n = 13), as well as cell lines established from tumors of 34 HNSCC patients. RESULTS: The results show specifically elevated gene expression of several proteinases, including MMP1, MMP3, MMP10, and MMP13 within tumor tissue and peritumoral adjacent tissue. In addition, the results identify several novel HNSCC-associated proteinases, including ADAM8, ADAM9, ADAM17, ADAM28, ADAMTS1, ADAMTS8, and ADAMTS15. There were also significant differences in proteinase expression based on clinical parameters, i.e., tumor location, grade, and local invasion. MMP13 expression was significantly higher in large (>4 cm) locally invasive tumors (P < 0.05). MMP9 expression was significantly decreased in tumors with regional metastasis, whereas increased expression of ADAM8 was noted in the metastatic tumors (P < 0.001 for both). CONCLUSIONS: These findings suggest the HNSCC degradome as a valuable source of diagnostic, predictive, and prognostic molecular markers for these malignant tumors.

Variant CD44 expression by human fibroblasts.

CD44 is a cell surface glycoprotein with roles in tumour invasion and metastasis. CD44 is variably spliced from ten variant exons and mis-splicing is a biomarker for detection of colon, urothelial and other carcinomas. Fibroblasts are normally considered to lack variant exons and thus should not generate false-positive signals. Transcription of variant exons by fibroblasts was investigated by exon-specific reverse transcription-polymerase chain reaction (RT-PCR) for variant exons v2-v10 using normal primary fibroblasts, immortalized and experimentally transformed fibroblasts. Flow cytometry, immunocytochemistry and Western blotting were used to determine expression. All types of fibroblasts, including normal primary culture fibroblasts, transcribed low levels of variant exon mRNA. Expression could not be detected by blotting or immunocytochemistry but flow cytometry revealed minor expression of some exons by all three types of cultured fibroblast. Fibroblasts do transcribe and express small amounts of variant exon CD44. This may need to be considered when using exon splicing as a biomarker for malignancy in clinical samples containing connective tissue.