Susceptibility of A. fumigatus‐specific T‐cell assays to pre‐analytic blood storage and PBMC cryopreservation greatly depends on readout platform and analytes

Mould‐specific T cells detectable by flow cytometry or ELISPOT were proposed as a novel biomarker in invasive aspergillosis. To define protocols facilitating sample shipment and longitudinal analysis, this study evaluated the susceptibility of different functional assays for A. fumigatus‐specific T‐cell quantification and characterisation to pre‐analytic delays. PBMCs from 6 healthy donors were analysed after immediate isolation, after 6 hours whole blood storage or after cryopreservation using 3 different common media. Functional responses to A. fumigatus lysate stimulation were comparatively assessed by flow cytometry, ELISPOT and 14‐plex cytokine assay. After 6 hours pre‐analytic storage, all functional assays showed reduced detection rates, higher coefficients of variation (CV) and widely varying individual recovery indices of specific T‐cell response. While cryopreservation resulted in sufficient yields and largely unaltered cellular composition, outcomes of functional readouts significantly differed from freshly processed samples. For CD154‐based flow cytometry, only cryopreservation in RPMI supplemented with autologous serum resulted in satisfactory detection rates and CVs. For ELISPOT and cytokine secretion assays, none of the cryopreservation protocols provided sufficient concordance with immediately processed samples. Even using the same readout platform, individual analytes widely varied in their susceptibility to cryopreservation, highlighting that distinct optimisation is required depending on the downstream assay.     

Monosomy 17 in potentially curable <Emphasis Type="Italic">HER2</Emphasis>-amplified breast cancer: prognostic and predictive impact

Purpose

HER2 copy number by fluorescence in situ hybridization (FISH) is typically reported relative to the centromere enumeration probe 17 (CEP17). HER2/CEP17 ratio could be impacted by alterations in the number of chromosome 17 copies. Monosomy of chromosome 17 (m17) is found in ~ 1900 cases of early-stage HER2-positive breast cancer annually in the United States; however, the efficacy of HER2-directed trastuzumab therapy in these patients is not well characterized. Here, we retrospectively identified HER2-amplified, stage I–III breast cancers with m17 and characterized the impact of trastuzumab treatment.

Methods

From January 1, 2000 to June 1, 2011, we identified 99 women with HER2-amplified m17 breast cancers, as defined by a CEP17 signal of < 1.5 per nucleus and a HER2/CEP17 ratio of ≥ 2.0.

Results

Most HER2-amplified m17 patients were treated with trastuzumab plus chemotherapy (51%, n = 50), whereas 31% (n = 31) received chemotherapy alone and 18% (n = 18) received no chemotherapy. The 4-year overall survival (OS) was superior with trastuzumab compared to chemotherapy alone or no chemotherapy (100 vs. 93 vs. 81%, respectively; p = 0.005). OS was not influenced by estrogen/progesterone-receptor (ER/PR) status, tumor stage, or degree of FISH positivity. A proportion of patients who would be considered HER2-negative by standard immunohistochemistry staging criteria (0–1+) were HER2 amplified by FISH.

Conclusions

In the largest series reported to date, patients with HER2-amplified m17 cancers treated with trastuzumab have outcomes comparable to patients from the large phase III adjuvant trastuzumab trials who were HER2-positive, supporting the critical role of HER2-directed therapy in this patient population.

     

Safety and Preliminary Efficacy of Vorinostat With R-EPOCH in High-risk HIV-associated Non-Hodgkin's Lymphoma (AMC-075)

Introduction

Vorinostat (VOR), a histone deacetylase inhibitor, enhances the anti-tumor effects of rituximab (R) and cytotoxic chemotherapy, induces viral lytic expression and cell killing in Epstein-Barr virus-positive (EBV⁺) or human herpesvirus-8-positive (HHV-8⁺) tumors, and reactivates latent human immunodeficiency virus (HIV) for possible eradication by combination antiretroviral therapy (cART).

Extracardiac <Superscript>18</Superscript>F-florbetapir imaging in patients with systemic amyloidosis: more than hearts and minds

Purpose

¹⁸F-Florbetapir has been reported to show cardiac uptake in patients with systemic light-chain amyloidosis (AL). This study systematically assessed uptake of ¹⁸F-florbetapir in patients with proven systemic amyloidosis at sites outside the heart.

Methods

Seventeen patients with proven cardiac amyloidosis underwent ¹⁸F-florbetapir PET/CT imaging, 15 with AL and 2 with transthyretin amyloidosis (ATTR). Three patients had repeat scans. All patients had protocolized assessment at the UK National Amyloidosis Centre including imaging with ¹²³I-serum amyloid P component (SAP). ¹⁸F-Florbetapir images were assessed for areas of increased tracer accumulation and time–uptake curves in terms of standardized uptake values (SUV_mean) were produced.

Results

All 17 patients showed ¹⁸F-florbetapir uptake at one or more extracardiac sites. Uptake was seen in the spleen in 6 patients (35%; 6 of 9, 67%, with splenic involvement on ¹²³I-SAP scintigraphy), in the fat in 11 (65%), in the tongue in 8 (47%), in the parotids in 8 (47%), in the masticatory muscles in 7 (41%), in the lungs in 3 (18%), and in the kidney in 2 (12%) on the late half-body images. The ¹⁸F-florbetapir spleen retention index (SRI) was calculated. SRI >0.045 had 100% sensitivity/sensitivity (in relation to ¹²³I-SAP splenic uptake, the current standard) in detecting splenic amyloid on dynamic imaging and a sensitivity of 66.7% and a specificity of 100% on the late half-body images. Intense lung uptake was seen in three patients, one of whom had lung interstitial infiltration suggestive of amyloid deposition on previous high-resolution CT. Repeat imaging showed a stable appearance in all three patients suggesting no early impact of treatment response.

Conclusion

¹⁸F-Florbetapir PET/CT is a promising tool for the detection of extracardiac sites of amyloid deposition. The combination of uptake in the heart and uptake in the spleen on ¹⁸F-florbetapir PET/CT, a hallmark of AL, suggests that this tracer holds promise as a screening tool for AL.

     

Comparison of the effects of sevoflurane and isoflurane on arterial oxygenation during one lung ventilation

We have compared the effects of sevoflurane and isoflurane on arterial oxygenation, heart rate and mean arterial pressure during one lung anaesthesia in a prospective, crossover study. We studied 28 patients undergoing oesophagogastrectomy, allocated alternatively to one of two groups. Patients in group I/S (n = 14) received 1 MAC (1.1%) of isoflurane in oxygen from induction until the end of 30 min of open chest one lung ventilation (OLV) in the lateral position. This was followed by 1 MAC (2.1%) of sevoflurane in oxygen for the next 30 min of OLV. Patients in group S/I (n = 14) received the two anaesthetic agents in the reverse order. We found no significant difference in arterial oxygenation, heart rate or mean arterial pressure between the two potent inhalation agents. In the subgroup of patients with pulmonary artery catheters (n = 12), we found a significant increase (P < 0.05) in derived shunt during sevoflurane anaesthesia. There was no significant difference in mixed venous saturation and cardiac output. We conclude that during one lung ventilation, the choice between sevoflurane and isoflurane did not significantly influence arterial oxygenation.      

Effects of systemic 3-nitropropionic acid-induced lesions of the dorsal striatum on cannabinoid and µ-opioid receptor binding in the basal ganglia

Systemic administration of 3-nitropropionic acid (3NPA) in experimental animals produces bilateral striatal lesions similar to those seen in Huntington’s disease (HD) caudate and putamen. [³H]-CP55,940 binding to cannabinoid receptors in human basal ganglia nuclei has been shown to be highly susceptible to the earliest pathological changes in the HD brain. In this study, to assess further the suitability of 3NPA-induced striatal lesions as a model for HD neuropathology, we examined the effects of striatal lesions induced by the systemic administration of 3NPA on the binding of [³H]-CP55,940 to pre- and postsynaptic cannabinoid receptors in striatum, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata and also the effect of 3NPA-induced striatal lesions on the binding of [³H]-DAMGO to μ-opioid receptors in striatal striosomes. Systemic administration of 3NPA induced bilateral and symmetrical lesions in dorsolateral striatum. Within the lesion core, [³H]-CP55,940 and [³H]-DAMGO binding density was reduced to background levels. Beyond the immediate borders of the central core of the 3NPA-induced lesion, striatal binding density was not significantly different from that measured in unlesioned rats. [³H]-CP55,940 binding in globus pallidus, entopeduncular nucleus and substantia nigra in 3NPA-lesioned rats was significantly reduced compared to controls, and the individual decreases were similar for each site. However, these reductions were statistically marginal. These data suggest that, while producing striatal lesions which bear some similarity to those seen in HD, the consequences of 3NPA for striatopallidal and striatonigral efferent projections do not reflect the reported neurodegenerative changes seen in the HD brain. Received: 18 November 1998 / Accepted: 12 July 1999     

Effects of indomethacin and polyunsaturated fatty acid diet on exercise-induced hypoxaemia in master athletes

We have previously reported a reduction in exercise-induced hypoxaemia following polyunsaturated fatty acid supplementation (PUFA). Although this might have been explained by increases in membrane fluidity, a clear explanation could not be provided due to potentially confounding influences of series-2 prosta- glandin mediated effects resulting from PUFA. In this investigation, ten master athletes [mean age 48.1 (SEM 6) years, maximal oxygen uptake (V˙O_{2 max} ) 3.39 (SEM 0.21) l?·?min^?1] completed a maximal cycling test (Ctrl) which was repeated after the administration of 150 mg of indomethacin to inhibit prostaglandin synthesis, both before and after 6 weeks of 3.66-g PUFA?·?day^?1. Cardiorespiratory parameters were obtained simultaneously with brachial arterial blood sampling for partial pressure of oxygen in arterial blood (P _aO₂), partial pressure of carbon dioxide in arterial blood (P _aCO₂), pH, oxygen saturation in arterial blood and lactate concentration determinations. A significant decrease in P _aO₂ (mmHg) from rest [93 (SEM 1.5)] was observed for exercise intensities of more than 40% V˙O_{2 max} in Ctrl reaching 75.9 (SEM 2.1) at V˙O_{2 max} . PUFA resulted in a 5.0 (SEM 0.68) mmHg upward shift (P?P _aO₂–oxygen uptake relationship, reducing the difference in partial pressure of oxygen between alveolar air and arterial blood (P _(A?a)O₂) at V˙O_{2 max} [Ctrl 36 (SEM 1.6) vs PUFA 33 (SEM 2.2) mmHg] while P _aCO₂, remained unchanged. Indomethacin had no effect on either P _aO₂, ideal partial pressure of oxygen in alveolar gas or P _(A?a)O₂ in either Ctrl or after PUFA. In contrast, the fall in pH was significantly reduced after indomethacin while V˙CO₂, P _aCO₂ and lactacidaemia remained unchanged. These observations confirm an effect of PUFA on exercise P _aO₂ behaviour which does not appear to be mediated by the influence of a series-2 prostaglandin.     

Exercise-induced hypoxaemia in master athletes: effects of a polyunsaturated fatty acid diet

Exercise-induced hypoxaemia (EIH) has been associated with an oxygen diffusion limitation. Because polyunsaturated fatty acids (PUFA) administration can modify cell membrane fluidity, we hypothesized that the importance of EIH could be reduced after a 6-week PUFA diet. Resting pulmonary functions and a maximal cycling test were performed before and after the diet, in eight master athletes [48 (SD 6 years)]. The partial pressure. of O₂ in arterial blood (PaO₂), alveolar ventilation ( ) and ideal alveolar-arterial oxygen partial pressure difference (P(A _i–a)O₂) were obtained at each exercise intensity. The extent of EIH at maximal exercise was significantly lower after PUFA [P_aO₂ –17.2 (SEM 1.9) vs –12.9 (SEM 2.2)]. Before PUFA, accounted for 50% of the variance in the fall inP(A _i–a) for intensities below 80% maximal oxygen uptake ( ) andP(A _i–a)O₂ for 60% between 70% and 100% . After PUFA, the reduction in EIH was highly correlated (r ² = 0.85;P < 0.001) to resulting changes inP(A_ii–a)O₂ and resting pulmonary diffusing capacity but not with changes in ideal alveolar partial pressure of oxygen. The improvement in EIH following PUFA could be related to an increase in alveolar-arterial oxygen conductance following improved pulmonary diffusion.