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The introduction of targeted therapy has revolutionized cancer treatment. Nonetheless, for this approach to succeed, it is crucial to identify the targets, particularly when activated, in tumor tissues. Phosphorylation is a posttranslational modification that causes activation of numerous oncogenic protein kinases and transcription regulators. Hence, phosphoproteins is a class of biomarkers that has therapeutic and prognostic implications directly relevant to cancer patients' management. Despite the progress in histopathology methodology, analysis of the expression of phosphoproteins in tumor tissues still represents a challenge owing to preanalytical and analytical factors that include antigen retrieval strategies. In this study, we tested the hypothesis that optimizing antigen retrieval methods will improve phosphoproteins unmasking and enhance their immunohistochemical staining signal. We screened 4 antigen retrieval methods by using antibodies specific for 3 oncogenic phosphoproteins to stain human lymphoma tumors that were developed in severe combined immunodeficiency mice and subsequently fixed in formalin for 2 years. Then, we used antibodies specific for 15 survival phosphoproteins to compare the most effective method identified from our screening experiment to the antigen retrieval method that is most commonly utilized. Using the antigen retrieval buffer Tris-EDTA at pH 9.0 and heating for 45 minutes at 97°C unmasked and significantly enhanced the staining of 9 of the 15 phosphoproteins (P0.0001). Our antigen retrieval approach is cost effective and feasible for clinical and research settings. We anticipate that combining this approach with the newly proposed methods to improve tissue fixation will further improve unmasking of phosphoproteins in human and animal tissues.

BACKGROUND AND AIMS Therapeutic, clinical trial entry and stratification decisions for hepatocellular carcinoma (HCC) are made based on prognostic assessments, using clinical staging systems based on small numbers of empirically selected variables that insufficiently account for differences in biological characteristics of individual patients' disease. APPROACH AND RESULTS We propose an approach for constructing risk scores from circulating biomarkers that produce a global biological characterization of individual patient's disease. Plasma samples were collected prospectively from 767 patients with HCC and 200 controls, and 317 proteins were quantified in a Clinical Laboratory Improvement Amendments-certified biomarker testing laboratory. We constructed a circulating biomarker aberration score for each patient, a score between 0 and 1 that measures the degree of aberration of his or her biomarker panel relative to normal, which we call HepatoScore. We used log-rank tests to assess its ability to substratify patients within existing staging systems/prognostic factors. To enhance clinical application, we constructed a single-sample score, HepatoScore-14, which requires only a subset of 14 representative proteins encompassing the global biological effects. Patients with HCC were split into three distinct groups (low, medium, and high HepatoScore) with vastly different prognoses (medial overall survival 38.2/18.3/7.1 months; P

We appreciate the thoughtful comments of Huo, Liu and Hsu. They mentioned their previous paper1 comparing 11 HCC staging systems, suggested from our C-index results "it should not be interpreted that the HepatoScore-14 significantly augment(s) the prognostic accuracy of the current systems," and wondered how our cohort characteristics impacted results. We make four comments in response.

B lymphoblastic leukemia/lymphoma (B-LBL) typically presents with symptoms related to anemia, thrombocytopenia, and neutropenia. CNS involvement is not uncommon during the disease course; however, it is rare as the initial presentation, making B-LBL with cranial nerve (CN) palsy a diagnostic challenge. Our objective is to show that B-LBL can present initially as CN palsy, and to share our approach to this challenge.

The plasma cell variant of Castleman's disease (PCD) may occur in a variety of clinical settings. One recently delineated type of PCD is caused by human herpesvirus 8 (HHV8) infection. Lymph nodes from 25 patients with PCD, including six HHV8+ and 19 HHV8- cases, were studied. Three patients with HHV8+ PCD were also infected with human immunodeficiency virus-1. Features common to all cases were interfollicular plasmacytosis and variably hyperplastic and regressed follicle germinal centers. Features associated only with HHV8+ PCD included follicle dissolution resulting from blurring of the mantle zone boundary (p = 0.0001), presence of atypical plasma cells and immunoblasts within these areas (p = 0.0006), and more prominent interfollicular vascular proliferation than in HHV8- PCD. HHV8+ cells were predominantly immunoblasts and small lymphocytes that were highly enriched in the mantle zones of altered follicles. These areas showed a predominance of plasmacytoid forms expressing lambda light chain in four of six cases. The extrafollicular fibroblastic network surrounding altered germinal centers demonstrated marked upregulation of low-affinity nerve growth factor receptor in five of five HHV8+ cases but in only two of 10 HHV8- cases. We conclude that HHV8+ PCD is distinctive histologically because of the accumulation of infected lymphocytes in the mantle zone leading to progressive dissolution of the germinal center and altered regulation of the surrounding stroma.

Methylnaltrexone (MNTX) is a quaternary derivative of naltrexone. It does not cross the blood-brain barrier and, thus, it reverses peripherally mediated effects of morphine without blocking its centrally located analgesic effects. The effects of MNTX on morphine-induced depression of hypoxic ventilatory response are unknown. We evaluated the efficacy of MNTX, compared with naloxone, in reversing this effect. On three sessions separated by a week, 10 healthy male volunteers received morphine, 0.125 mg/kg, as a bolus at 20 min after completing a control hypoxic ventilatory challenge. At 60 min, naloxone, 5 micrograms/kg, MNTX, 0.3 mg/kg, or placebo was administered in a randomized double-blind order. Four isocapnic hypoxic ventilatory challenges were conducted: 0 min (control), 40 min (postmorphine), and 80 and 120 min (postreversal) and the hypoxic respiratory responses were recorded. Morphine administration was associated with a significant depression in hypoxic responses: The slope of the response (L/min/Spo2) and the predicted ventilation at 80% O2 saturation (VE80) (L/min) decreased significantly in the three sessions (P < 0.05). Naloxone injection reversed the respiratory depression at 80 min (85% of the control value of the slope and 89% of VE80), whereas MNTX and placebo did not. At 120 min, the slope (69%) and VE80 (80%) after naloxone administration were not significantly different from control. MNTX slope (69%) was not statistically different from the control, whereas VE80 (70%) was still depressed (P < 0.05). Placebo slope and VE80, at 120 min, remained lower than the control (P < 0.05). These data show that MNTX is not as effective as naloxone for reversal of morphine-mediated depression of respiration during acute hypoxia.