Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality.

BACKGROUND: We have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features. RESULTS: Here we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue. CONCLUSIONS: In summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.

Composite EBV negative peripheral T-cell lymphoma and diffuse large B-cell lymphoma involving the ileum: a case report and a systematic review of the literature.

We report a case of an intestinal peripheral T-cell lymphoma (PTCL) with a concurrent diffuse large B-cell lymphoma (DLBL) involving the ileum and a regional lymph node. The patient presented with an abdominal mass. The terminal ileum showed a diffuse and monotonous population of small CD3-positive T cells. The T-cell receptor gamma (TCRgamma) gene was rearranged by PCR while the immunoglobulin heavy chain (IgH) gene was not. A separate section of the ileum showed a colliding large B-cell proliferation. The regional lymph node showed a diffuse proliferation of large centroblasts positive for CD20 and CD79a admixed with small T cells and showed a rearranged IgH receptor gene without evidence of a clonally rearranged TCRgamma gene. Both the PTCL and DLBL components were negative for EBV. A review and analysis of the pertinent literature describing composite T- and B-cell lymphomas is performed and reported.

Development and clinical validation of an in situ biopsy-based multimarker assay for risk stratification in prostate cancer.

PURPOSE: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. EXPERIMENTAL DESIGN: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish "favorable" versus "nonfavorable" pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). RESULTS: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on "false-negative" and "false-positive" rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non-GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). CONCLUSIONS: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy.

Separation of IgG and IgM from albumin in citrated human plasma using electrodialysis and metal ion affinity precipitation.

Plasmapheresis is the process by which plasma is separated from whole blood for therapeutic purposes. The primary reason to perform plasmapheresis is to remove immunoglobulins and not to necessarily remove the remainder of the plasma proteins. Nevertheless, most plasmapheresis is performed by centrifugal separation to remove bulk plasma. We have investigated whether electrodialysis and metal ion affinity precipitation can be used to selectively remove immunoglobulins from citrated plasma and potentially serve as an adjunctive technique to centrifugal plasmapheresis. Using a commercial electrodialysis device, we have desalted citrated plasma to separate immunoglobulins from albumin, and have determined the effects of pH adjustment and addition of zinc acetate for concomitant metal ion affinity precipitation. With desalted pH adjusted citrated plasma containing 1.5 mM zinc acetate, we achieved more than 80% recovery of albumin with removal of almost 60% of the immunoglobulin (Ig)G. Almost 80% of polyclonal IgM and 90% of monoclonal IgM was also removed. IgA was not effectively removed under any of the conditions tested. Selective precipitation with electrodialysis and zinc acetate precipitation appears to be an effective technique for the separation of IgG and IgM from albumin in citrated plasma.

Chronic in vivo testing of the Penn State infant ventricular assist device.

The Penn State Infant Ventricular Assist Device (VAD) is a 12-14 ml stroke volume pneumatically actuated pump, with custom Björk-Shiley monostrut valves, developed under the National Heart, Lung, and Blood Institute Pediatric Circulatory Support program. In this report, we describe the seven most recent chronic animal studies of the Infant VAD in the juvenile ovine model, with a mean body weight of 23.5 ± 4.1 kg. The goal of 4-6 weeks survival was achieved in five of seven studies, with support duration ranging from 5 to 41 days; mean 26.1 days. Anticoagulation was accomplished using unfractionated heparin, and study animals were divided into two protocol groups: the first based on a target activated partial thromboplastin time of 1.5-2 times normal, and a second group using a target thromboelastography R-time of two times normal. The second group required significantly less heparin, which was verified by barely detectable heparin activity (anti-Xa). In both groups, there was no evidence of thromboembolism except in one animal with a chronic infection and fever. Device thrombi were minimal and were further reduced by introduction of the custom valve. These results are consistent with results of adult VAD testing in animals and are encouraging given the extremely low levels of anticoagulation in the second group.

The effect of catheter to vein ratio on blood flow rates in a simulated model of peripherally inserted central venous catheters.

BACKGROUND: Catheter-related thrombosis is a common complication in all anatomic sites, especially when smaller veins of the upper extremity are considered. It is presumed that the presence of a catheter within the lumen of a vein will decrease flow and potentially create stasis, and clinical data suggest that the size of the catheter impacts thrombosis rates. We sought to determine, both mathematically and experimentally, the impact of catheters on fluid flow rates. METHODS: We used fluid mechanics to calculate relative flow rates as a function of the ratio of the catheter to vein diameters. We also measured the flow rate of a blood analyte solution in an annular flow model using diameters that simulate the size of upper extremity veins and commonly used peripherally inserted central catheters (PICCs). RESULTS: We compared each of the derived relative flow rates to the experimentally determined ones for three cylinder sizes and found a correlation of r(2) = 0.90. We also confirmed that the decrease in fluid flow rate with each successive catheter size is statistically significant (P < .0001). CONCLUSIONS: Our results demonstrate that fluid flow is dramatically decreased by the insertion of a centrally located obstruction. Assuming that blood flow in veins behaves in a similar manner to our models, PICCs, in particular, may substantially decrease venous flow rates by as much as 93%.

Oxygen saturation and hemoglobin A content in patients with sickle cell disease undergoing erythrocytapheresis.

Severe hypoxia occurs in patients with acute chest syndrome, and erythrocytapheresis has been shown to improve oxygenation. Patients with sickle cell anemia also have decreased baseline oxygen saturation values, but the effect of erythrocytapheresis on steady-state oxygenation has not been well studied. We investigated the changes in oxygen saturation versus hematocrit, fraction of hemoglobin A, and transfusion volume during 71 prophylactic erythrocytapheresis procedures performed in 5 stable patients with sickle cell anemia. Each patient had a history of either acute chest syndrome or stroke, but no serious events occurred while enrolled in the chronic exchange program. The oxygen saturation improved from 1% to 6% during erythrocytapheresis in each of our patients (p < 0.001) regardless of preprocedure saturation level or total hematocrit. We have shown that decreased baseline oxygen saturation in sickle cell disease is related to abnormal hemoglobin S levels, and oxygen saturation can be improved with erythrocytapheresis, independent of any change in the total hematocrit.

Coagulant stability and sterility of thawed S/D-treated plasma.

BACKGROUND: Units of frozen S/D-treated plasma (SDP) must be transfused within 24 hours after thawing. To avoid waste, an attempt was made to determine how long SDP could be therapeutically effective after thawing and storing it at 20 degrees C. STUDY DESIGN AND METHODS: The microbiologic safety and the activity of labile coagulation factors were evaluated in units stored at 20 degrees C of thawed SDP units and FFP within 24 hours of collection (FFP24). Five SDP and FFP24 samples of each ABO blood group were cultured and assayed for coagulation factors daily over 5 days. Assays included FV, FVII, FVIIa, FVIII, F IX, FXI, protein S, antiplasmin, fibrinogen, prothrombin times (PTs), and activated partial thromboplastin times (aPTTs). RESULTS: None of the 80 bacterial cultures demonstrated growth under either aerobic or anaerobic conditions. FV, FVIII, F IX, FXI, fibrinogen, and the aPTT appeared to be stable in both thawed FFP24 and SDP. The PT increased slightly in thawed FFP24 and insignificantly in SDP. FVII decreased slightly in FFP24 but remained in the normal range, and FVIIa was low and constant. FVII was increased in SDP and FVIIa was markedly increased. Protein S decreased from initial normal values in FFP24 to very low values. Protein S was very low immediately after thawing in the SDP and continued to decline. Antiplasmin was normal and stable in thawed FFP24 but was low in SDP and remained constant after thawing. CONCLUSION: Sterile SDP that is stored at 20 degrees C provides sufficient coagulant activity of labile FV and FVIII to transfuse it for up to 5 days after thaw. Caution is warranted by decreases in Protein S and antiplasmin, clinical evidence of coagulopathy in some recipients of SDP, and a recent manufacturer's warning.

Durable response to combination therapy including staphylococcal protein A immunoadsorption in life-threatening refractory autoimmune hemolysis.

BACKGROUND: Few therapeutic options are available for severe, life-threatening, refractory autoimmune hemolytic anemia. CASE REPORT: A 53-year-old 110-kg man was seen with acute onset of symptomatic severe anemia with syncope, unstable angina, and jaundice. His nadir Hct was 8.3 percent with a peak total bilirubin of 44 mg per dL. The DAT was positive but the IAT was negative. Elution studies demonstrated an IgG pan-agglutinin antibody reactive at 37 degrees C. Treatment with high-dose corticosteroids and IVIG was instituted. An accessory spleen measuring 2 cm was identified and surgically removed, but the patient continued to have intense hemolysis. Cyclophosphamide at 200 mg per day was started. Apheresis with a staphylococcal protein A immunoadsorption column (Prosorba, Cypress Bioscience, Inc.) was initiated on Day 18 and was performed twice weekly for a total of six treatments. Cyclophosphamide was continued for a total of 14 days. His transfusion requirement ceased by the third immunoadsorption treatment. Forty units of RBCs were required over 23 days in an attempt to maintain a Hct greater than or equal to 15 percent. CONCLUSION: Refractory autoimmune hemolysis can be a life-threatening event. The patient did not achieve a response until after several different therapeutic modalities were instituted, including plasmapheresis with a staphylococcal protein A column (Prosorba). A complete response continues to be durable for more than 1 year after therapy.

Favorable outcome after infusion of coagulase-negative staphylococci-contaminated peripheral blood hematopoietic cells for autologous transplantation.

Bacterial contamination of peripheral blood hematopoietic cells collected for autologous bone marrow transplantation occurs sporadically. Although transfusion of contaminated hematopoietic cells without adverse clinical sequelae has been reported, detailed guidelines for transfusing cells with contamination are not available. We report a case of autologous hematopoietic cell transplantation that necessitated using multiple aliquots of peripheral blood hematopoietic cells known to be contaminated with coagulase-negative Staphylococcus bacteria. Prophylactic intravenous antibiotic therapy was given with the infusion of contaminated hematopoietic cells. The patient had positive results on a blood culture, but engraftment was successful, and serious adverse effects did not occur. With appropriate microbial identification and prophylactic antibiotic therapy, contaminated hematopoietic products can be safely infused when necessary with a good clinical outcome.