Research Foundation results

Determination of high-risk HPV status of head and neck squamous cell carcinoma using the Roche cobas HPV test on cytologic specimens and acellular supernatant fluid.

High-risk human papillomavirus-positive (hrHPV+) oropharyngeal squamous cell carcinomas comprise a subset of head and neck squamous cell carcinomas (HNSCCs) with a distinct biology and prognosis. Commonly, the diagnosis of HNSCC is rendered on fine-needle aspiration (FNA). Because cell blocks may be insufficient for determining HPV status using microscopy-based techniques, the ability of liquid-based assays was examined in the current study.The performance of the Roche cobas 4800 platform was evaluated on the FNA material from the cell pellet and corresponding cell-free supernatant fluid specimens of primary and metastatic HNSCCs. These results were compared with the p16 immunostain result from the histologic material obtained from the same patient. Discrepant cases were adjudicated using hrHPV RNA in situ hybridization.A total of 41 samples (23 primary tumors and 18 lymph node metastases) were acquired from 34 patients with HNSCC. Primary tumors included the oropharynx (20 samples), oral cavity (13 samples), larynx (3 samples), and skin (3 samples). In 2 cases, a primary tumor could not be identified. Twenty-three samples (56%) were found to be p16 positive by immunohistochemistry. Twenty-two samples were found to be positive on cobas hrHPV testing from both cell pellet and cell-free supernatant fluid. Two cell-free supernatant fluid specimens yielded indeterminate cobas results. At the time additional hrHPV RNA in situ hybridization analysis was performed, one cobas-positive cell pellet was deemed to be a false-positive result. The sensitivity of the cobas assay was 100% for pellet material and cell-free supernatant fluid, with specificities of 94.7% and 100%, respectively. cobas hrHPV testing of HNSCC specimens demonstrated high concordance with p16 immunohistochemistry on the corresponding cell block and/or tissue specimen. Using the cell-free supernatant fluid in this platform could provide accurate HPV results while conserving material for cytomorphologic analyses.

Serum uric acid a depression biomarker.

We aimed to investigate the difference in serum uric acid(SUA)levels between subtypes of depression and normal population, and whether SUA can be used to identify bipolar disorder depressive episode and major depressive disorder and predict the length of hospital stay.1543 depression patients and 1515 healthy controls were obtained according to the entry and exclusion criteria from one mental health center of a tertiary hospital in southwestern China. The diagnosis and classification of depression was in accordance with ICD-10. The SUA value was derived from fasting plasma samples analysis. The level of SUA of all the participants was quantified using Roche cobas8000-c702-MSB automatic biochemical analyzer. Data were analyzed by SPSS18.0 statistical software package.

Overall, the level of SUA in patients with depression was lower than that in normal control. Specifically, males’ SUA levels were in the interval of [240, 323.3) and [323.3, 406.6), and women were in the [160, 233.3] levels.

The SUA level of bipolar disorder depressive episode was higher compared to major depressive disorder level. Interestingly, male patients who were hospitalized for two weeks had higher SUA than those who were hospitalized for three weeks or four weeks.Our results suggest that the length of hospital stay may be associated with SUA, and when it is difficult to make a differential diagnosis of bipolar disorder depressive episode and major depressive disorder, the level of SUA may be considered. The adjustment of SUA as a method for treating depression needs to be carefully assessed.

Age-specific reference intervals for plasma free thyroxine and thyroid stimulating hormone in term neonates during the first two weeks of life.

Background Congenital hypothyroidism (CH) is a common and preventable cause of mental retardation, which is detected in many neonatal screening programs. Upon suspicion of CH, plasma free thyroxine (FT4) and thyroid stimulating hormone (TSH) concentrations are measured. CH can be of thyroidal or central origin (CH-T and CH-C, respectively). While CH-T diagnosis is based on an elevated TSH with a low FT4, CH-C diagnosis is based on a low FT4 without a clearly elevated TSH.

Currently, reliable neonatal reference intervals (RIs) for plasma FT4 and TSH are lacking. Age-specific RIs would greatly improve the diagnostic process for CH, especially for CH-C. Our aim was to establish neonatal RIs for plasma FT4 and TSH in term neonates at day 3-7 (t=1) and day 13-15 (t=2). The study was particularly designed to provide a reliable FT4 lower limit of the RI to facilitate the diagnosis of CH-C. In the Netherlands, neonates are screened at day 3-7 of life.

After a screening result suggestive for CH-C, pediatric consultation takes place on average at day 14. Thus, the time points were chosen accordingly. Methods Venous blood was collected from 120 healthy neonates at each time point (94 participants provided blood samples at two time points; 52 participants provided a sample at t=1 or t=2).

FT4 and TSH were measured using an immunoassay (Cobas, Roche Diagnostics and Maglumi 800, Genprice).

RIs were calculated using the 95% confidence interval for normally distributed data, and the non-parametric percentile method if data were not normally distributed. Results From 146 participants (49% female) ≥1 measurement was available. 95% RIs for FT4 were 20.5-37.1 pmol/L (day 3-7) and 15.3-26.5 pmol/L (day 13-15). 95% RIs for TSH were 1.0-8.4 mU/L (day 3-7) and 1.4-8.6 mU/L (day 13-15). Conclusions Our results indicate a FT4 lower limit of the RI of 20.5 pmol/L at day 3-7, and 15.3 pmol/L at day 13-15. These lower limits are considerably higher than this assay’s lower limit of the adult RI for FT4. In case CH is suspected, we recommend measuring FT4 and TSH using an assay with an established neonatal RI, taking into account the child’s age in days.

Immunoassay interference on thyroid functions tests during treatment with nivolumab.

Immune checkpoint inhibitors (ICI) are associated to several endocrine side effects. In particular, the use of PD-1/PD-L1 inhibitors is related to a higher incidence of thyroid dysfunction.A 85 years-old patient, diagnosed with a metastatic melanoma treated with nivolumab, presented to our hospital with severe ICI-related thyrotoxicosis. The diagnosis was complicated by a biochemical interference on thyroid hormones assay, probably induced by nivolumab.Baseline laboratory examination conducted before the onset of anticancer therapy showed normal thyroid function test (TFTs).

A few days after receiving the second nivolumab administration, the patient developed a severe thyrotoxicosis. According to destructive thyroiditis, in a short period thyroid-stimulating hormone (TSH) levels normalized and rapidly increased, but free thyroxine (FT4) levels were inappropriately elevated and did not decrease as expected. The sample was processed by using a Siemens Centaur® immunoassay. We reanalyzed the same sample at another laboratory and with a different immunoassay method (Roche Elecsys®). The results obtained from this assay confirmed severe hypothyroidism with appropriately low FT4 levels. We suspected a possible nivolumab-associated interference on the FT4 assay. Therefore, we subjected the same sample a polyethylene glycol (PEG) 6000 precipitation, a simple method for the removal of macromolecules, before assaying for FT4 levels. The evaluation of the post-PEG-precipitation sample (Siemens Centaur® immunoassay) revealed appropriately low FT4 levels. The patient was started on levothyroxine therapy, with monthly TFT monitoring using the Roche immunoassay. Approximately 9 months after starting nivolumab therapy, the patient was advised treatment cessation.

A month later, the TFTs were retested on a Siemens Centaur® immunoassay, and appropriate FT4 levels were observed in accordance with normal TSH levels on adequate levothyroxine replacement therapy.We report a possible novel nivolumab-induced biochemical interference on assays of FT4 levels. The hypothesis of a biochemical drug-induced interference is further supported by the disappearance of the interference after the withdrawal of nivolumab. Further studies are needed to prove the biochemical mechanisms of this interference.

Age, absolute CD4 count, and CD4 percentage in relation to HPV infection and the stage of cervical disease in HIV-1-positive women.

A subgroup of women who are co-infected with human immunodeficiency virus type 1 (HIV-1) and human papillomavirus (HPV), progress rapidly to cervical disease. We characterized HPV genotypes within cervical tumor biopsies, assessed the relationships of cervical disease stage with age, HIV-1 status, absolute CD4 count, and CD4 percentage, and identified the predictive power of these variables for cervical disease stage in a cohort of South African women.We recruited 181 women who were histologically diagnosed with cervical disease; 87 were HIV-1-positive and 94 were HIV-1-seronegative. Colposcopy-directed tumor biopsies were confirmed by histology and used for genomic DNA extraction. The Roche Linear Array HPV genotyping test was used for HPV genotyping. Peripheral whole blood was used for HIV-1 rapid testing. Fully automated FC500MPL/CellMek with PanLeucogate (PLG) was used to determine absolute CD4 count, CD4 percentage, and CD45 count. Chi-squared test, a logistic regression model, parametric Pearson correlation, and ROC curves were used for statistical analyses. We used the Benjamini-Horchberg test to control for false discovery rate (FDR, q-value). All tests were significant when both P and q were <.05.Age was a significant predictor for invasive cervical cancer (ICC) in both HIV-1-seronegative (P < .0001, q < 0.0001) and HIV-1-positive women (P = .0003, q = 0.0003). Sixty eight percent (59/87) of HIV-1-positive women with different stages of cervical disease presented with a CD4 percentage equal or less than 28%, and a median absolute CD4 count of 400 cells/μl (IQR 300-500 cells/μl). Of the HIV-1-positive women, 75% (30/40) with ICC, possessed ≤28% CD4 cells vs 25% (10/40) who possessed >28% CD4 cells (both P < .001, q < 0.001). Furthermore, 70% (28/40) of women with ICC possessed CD4 count >350 compared to 30% (12/40) who possessed CD4 count ≤ 350 (both P < .001, q < 0.001).Age is an independent predictor for ICC. In turn, development of ICC in HIV-1-positive women is independent of the host CD4 cells and associates with low CD4 percentage regardless of absolute CD4 count that falls within the normal range. Thus, using CD4 percentage may add a better prognostic indicator of cervical disease stage than absolute CD4 count alone.

Assessment of biotin interference in thyroid function tests.

The aim of the study was to systematically characterize the interference of biotin on thyroid function tests and biotin washout periods.Ten healthy adults were recruited with administration of 5 and 10 mg/d biotin for 7 days. Analyte concentrations of thyroid function tests were measured at baseline prior to starting biotin and from 2 hours to 2 days after withdrawal of 5 and 10 mg/d biotin. The outcomes were compared the baseline with the several points after taking biotin at Roche cobas e602, Beckman UniCel DxI 800, and Abbott Architect 2000 immunoassay platforms, respectively.Ingesting 5 or 10 mg/d of biotin for 7 days could produce positive or negative interference among the thyroid function tests at Roche cobas e602 and Beckman UniCel DxI 800 systems, but no interference on Abbott Architect 2000. Interference duration of 5 mg/d biotin for Roche cobas e602 and Beckman UniCel DxI 800 of thyroid function tests lasted for 8 hours, while 10 mg/d biotin interfered with Roche cobas e602 or Beckman UniCel DxI 800 for 1 day or 2 days.This study provides valuable guidance on biotin washout periods at doses common in over-the-counter supplements necessary to avoid false assay results.Trial registration: ChiCTR1800020472.

Sex-Specific 99th Percentile Upper Reference Limits for High Sensitivity Cardiac Troponin Assays Derived Using a Universal Sample Bank.

How to select healthy reference subjects in deriving 99th percentiles for cardiac troponin assays still needs to be clarified. To assist with global implementation of high sensitivity (hs)-cardiac troponin (cTn) I and hs-cTnT assays in clinical practice, we determined overall and sex-specific 99th percentiles in 9 hs-cTnI and 3 hs-cTnT assays using a universal sample bank (USB).The Universal Sample Bank (USB) comprised healthy subjects, 426 men and 417 women, screened using a health questionnaire. Hemoglobin A1c (>URL 6.5%), NT-proBNP (>URL 125 ng/L) and eGFR (<60 mL/min), were used as surrogate biomarker exclusion criteria along with statin use. 99th percentiles were determined by nonparametric, Harrell–Davis bootstrap, and robust methods.Subjects were ages 19 to 91 years, Caucasian 58%, African American 27%, Pacific Islander/Asian 11%, other 4%, Hispanic 8%, and non-Hispanic 92%. The overall and sex-specific 99th percentiles for all assays, before and after exclusions (n = 694), were influenced by the statistical method used, with substantial differences noted between and within both hs-cTnI and hs-cTnT assays. Men had higher 99th percentiles (ng/L) than women. The Roche cTnT and Beckman and Abbott cTnI assays (after exclusions) did not measure cTn values at ≥ the limit of detection in ≥50% women.Our findings have important clinical implications in that sex-specific 99th percentiles varied according to the statistical method and hs-cTn assay used, not all assays provided a high enough percentage of measurable concentrations in women to qualify as a hs-assay, and the surrogate exclusion criteria used to define normality tended to lower the 99th percentiles.

A Comparison of Different Valgancyclovir Formulations in the Universal 6-Month Prophylaxis Against CMV Infection in Renal Transplant Recipients: A Randomized Single-Centre Study.

Cytomegalovirus (CMV) is the most common opportunistic infective pathogen in kidney transplant recipients. Valganciclovir (VAL) is commonly used for prophylaxis, especially in high-risk recipients. Generic VAL formulations have become available, but the data about their safety and efficacy are lacking.Consecutive de novo kidney transplant patients were randomized to generic VAL Valganciklovir Teva® (VT group)(24 patients) or Alvanocyte® (A group), Alvogen (19 patients) or to Valcyte® (V group), Roche (23 patients) in a 18-month open-label study. Universal prophylaxis was used for 6 months after the transplantation. CMV DNA levels were measured at 1,3,6,9,12 and 18 months after the transplantation. All positive measurements of CMV DNA were recorded.Groups did not differ regarding the clinical characteristics or the risk for developing CMV infection in the post-transplant period. CMV replications were most common at 9 months after the transplantation with rates of 9% for the V, 13% for the VT and 26% for the A group (p=0.26). At 12 months, positive CMV DNA was recorded in 22%, 8% and 11 % of patients taking V, VT and A, respectively (p=0.37). Rates of biopsy-proven acute rejection, adverse events, and serious adverse events were similar for all formulations. Lymphocele occurred most commonly in the V group (35%) compared to 17% in VT and 17% in the A group (p=0.23). One patient from each of the A and VT groups developed CMV disease. Additionally, they were the only two patients with CMV DNA copies above 656 IU/ml. Glomerular filtration rates were similar in all groups at all time points, while proteinuria was significantly higher at 12 months in patients who received V 0.32 g/day (0.18 – 0.42), compared to patients on VT 0.2 (0.1 – 0.2), or A 0.2 (0.2 – 0.3), p=0.04.Valgancyclovir efficacy and safety in this limited data set is similar with early administration of V, VT and A after kidney transplantation. Additional studies aimed at elucidating the effectiveness of this treatment regimen in patients who are at high risk for developing CMV infection are necessary to draw further conclusions.

Bile biochemistry following liver reperfusion in the recipient and its association with cholangiopathy.

Cholangiocytes secrete bicarbonate and absorb glucose, producing bile with alkaline pH and low glucose content. These functions of cholangiocytes have been suggested as a marker of bile duct viability during normothermic ex situ liver perfusion (NESLiP), and are now monitored routinely post reperfusion in our centre. In this study, we reviewed the composition of bile immediately after reperfusion in liver transplant recipients to determine normal post-transplant parameters and the predictive value of bile biochemistry for the later development of cholangiopathy. After reperfusion of the liver graft, a cannula was placed in the bile duct to collect bile over a median 44 minute time period. The bile produced was analysed using a point of care blood gas analyser (Roche Cobas b221). 100 liver transplants (35 donation after circulatory death [DCD] and 65 donation after brain death [DBD]) were studied. Median bile pH was 7.82 (Interquartile range [IQR] 7.67 -7.98), median bile glucose 2.1 (1.4 – 3.7) mmol/L, median blood and bile pH difference 0.5 (0.37 – 0.62) and the median blood and bile glucose difference 7.1 (5.6 – 9.1) mmol/L. 12 recipients developed cholangiopathy over a median follow up of 15 months (IQR 11 – 20). Bile sodium (142 vs 147 mmol/L; p=0.02) and blood-bile glucose concentration differences (5.2 vs 7.6; p=0.001) were significantly lower and associated with ischaemic cholangiopathy. In conclusion, bile biochemistry may provide useful insights into cholangiocyte function, and hence bile duct viability. Our results suggest bile glucose is the most sensitive predictor of cholangiopathy.

The Effectiveness and Value of Siponimod for Secondary Progressive Multiple Sclerosis.

Funding for this summary was contributed by Arnold Ventures, Commonwealth Fund, California Health Care Foundation, National Institute for Health Care Management (NIHCM), New England States Consortium Systems Organization, Blue Cross Blue Shield of Massachusetts, Harvard Pilgrim Health Care, Kaiser Foundation Health Plan, and Partners HealthCare to the Institute for Clinical and Economic Review (ICER), an independent organization that evaluates the evidence on the value of health care interventions. ICER’s annual policy summit is supported by dues from Aetna, America’s Health Insurance Plans, Anthem, Allergan, Alnylam, AstraZeneca, Biogen, Blue Shield of CA, Cambia Health Services, CVS, Editas, Express Scripts, Genentech/Roche, GlaxoSmithKline, Harvard Pilgrim, Health Care Service Corporation, Health Partners, Johnson & Johnson (Janssen), Kaiser Permanente, LEO Pharma, Mallinckrodt, Merck, Novartis, National Pharmaceutical Council, Premera, Prime Therapeutics, Regeneron, Sanofi, Spark Therapeutics, and United Healthcare. Synnott and Pearson are employed by ICER. Bloudek and Carlson report a research agreement between the University of Washington and ICER; Bloudek reports consulting fees from Allergan, Seattle Genetics, Dermira, Sunovion, TerSera Therapeutics, Cook Regentech, and Mallinckrodt Pharmaceuticals; and Carlson reports personal fees from Bayer, unrelated to this report. Sharaf reports consulting fees from ICER.

Acute macular neuroretinopathy with coexistent central retinal vein occlusion as the presenting feature in intraocular tuberculosis.

To report a case of intraocular tuberculosis presenting as acute macular neuroretinopathy and central retinal vein occlusion.A 29-year-old man presented to the retina clinic with complaints of sudden blurring of vision in the left eye of 3 days duration. His visual acuity was 6/6 and 6/18 in the right and left eye, respectively. Fundus examination of the left eye showed features of central retinal vein occlusion. OCT showed features of type 2 acute macular neuroretinopathy (AMN) as well. Over a period of 2 weeks, the patient developed choroidal granulomas with overlying retinal elevation and peripapillary choroidal neovascular membrane and retinal granuloma. Mantoux test and HRCT chest confirmed the diagnosis of pulmonary tuberculosis.The patient was treated with a course of antitubercular therapy, oral corticosteroids and a single dose of intravitreal anti-vascular endothelial growth factor (1.25 mg/0.05 ml Bevacizumab, Roche Pharma) injection. After 6 months of therapy with ATT and tapering course of oral steroids, there was a complete resolution of all clinical signs including the choroidal granuloma with an improvement in visual acuity to 6/6.Acute macular neuroretinopathy can complicate intraocular TB. Tuberculosis should be kept as one of the differential diagnosis in patients with AMN. Prognosis is generally good in patients of ocular TB presenting with retinal vascular occlusions.

Dynamic Contrast-Enhanced MRI Confirms Rapid And Sustained Improvement Of Rheumatoid Arthritis Induced By Tocilizumab Treatment: An Italian Multicentre Study.

This open-label study evaluated the effects of combined tocilizumab (TCZ) and disease-modifying antirheumatic drugs (DMARDs) on magnetic resonance imaging (MRI) changes in synovial membrane enhancement, bone marrow edema (BME), and erosions in the wrist and hand joints of rheumatoid arthritis (RA) patients inadequately responding to DMARDs alone.The efficacy of intravenous TCZ 8 mg/kg administered every four weeks for 48 weeks was evaluated on six occasions. The primary endpoints were the changes in the extent and degree of wrist synovitis as measured using the RA MRI Score (RAMRIS) and dynamic, gadolinium-enhanced 0.2T MRI (DCE-MRI). A number of different parameters of DCE-MRI were evaluated.


Fifty-eight patients were treated, eight of whom (13.8%) discontinued the study prematurely. The mean RAMRIS significantly decreased after two weeks and the decrease was maintained for up to 48 weeks. By week 4, the mean RAMRIS synovitis score had significantly decreased from baseline (-0.804±1.575; p=0.018), but not the mean early enhancement (REE) or relative enhancement (RE). However, there were significant decreases in RE at week 24, in REE and Ntotal (total number of enhancing voxels)IRE (initial rate of enhancement) at weeks 12, 24 and 48, and in NtotalME (maximal enhancement) at weeks 24 and 48. Mean BME decreased from baseline to week 48, and bone erosions did not progress. The patients’ clinical parameters significantly improved from baseline until week 48.
TCZ in combination with DMARDs improved wrist synovitis, BME and clinical parameters, without any progression in bone erosions. The RAMRIS for synovitis rapidly improved from as early as two weeks after the first TCZ infusion. (Funded by F. Hoffmann-La Roche; ACTRACE EudraCT No. 2009 012185-32).

The Value of a Patient-Level Modeling Approach and Need for Better Reporting in Economic Evaluations of Osteoporosis.

No funding was involved in the writing of this letter.

Outside of the submitted work, Hiligsmann has received research grants through institution from:

Amgen, Radius Health, UCB, and Teva/Theramex. Reginster has received research grants and/or consulting fees from Servier, Novartis, Negma, Lilly, Wyeth, Amgen, GlaxoSmithKline, Roche, Merckle, Nycomed-Takeda, NPS, IBSA Genevrier, Theramex, UCB, Asahi Kasei, Endocyte, Merck Sharp and Dohme, Rottapharm, Teijin, Teva, Analis, NovoNordisk, Ebewee Pharma, Zodiac, Danone, Will Pharma, Meda, Bristol Myers Squibb, Pfizer, Organon, Therabel, Boehringer, Chiltern, and Galapagos.

Silverman has received grant support from Amgen, Radius, and Lilly; consulting fees from Amgen and Radius; has served on scientific advisory boards for Lilly and Amgen; and has served on speakers bureaus for Amgen, Lilly, and Radius.

Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling.