Inflammatory breast cancer defined: proposed common diagnostic criteria to guide treatment and research.

PURPOSE: Inflammatory breast cancer is a deadly and aggressive type of breast cancer. A key challenge relates to the need for a more detailed, formal, objective definition of IBC, the lack of which compromises clinical care, hampers the conduct of clinical trials, and hinders the search for IBC-specific biomarkers and treatments because of the heterogeneity of patients considered to have IBC. METHODS: Susan G. Komen, the Inflammatory Breast Cancer Research Foundation, and the Milburn Foundation convened patient advocates, clinicians, and researchers to review the state of IBC and to propose initiatives to advance the field. After literature review of the defining clinical, pathologic, and imaging characteristics of IBC, the experts developed a novel quantitative scoring system for diagnosis. RESULTS: The experts identified through consensus several "defining characteristics" of IBC, including factors related to timing of onset and specific symptoms. These reflect common pathophysiologic changes, sometimes detectable on biopsy in the form of dermal lymphovascular tumor emboli and often reflected in imaging findings. Based on the importance and extent of these characteristics, the experts developed a scoring scale that yields a continuous score from 0 to 48 and proposed cut-points for categorization that can be tested in subsequent validation studies. CONCLUSION: To move beyond subjective 'clinical diagnosis' of IBC, we propose a quantitative scoring system to define IBC, based on clinical, pathologic, and imaging features. This system is intended to predict outcome and biology, guide treatment decisions and inclusion in clinical trials, and increase diagnostic accuracy to aid basic research; future validation studies are necessary to evaluate its performance.

Decoration of trastuzumab with short oligonucleotides: synthesis and detailed characterization.

Trastuzumab (Herceptin®) is an FDA-approved therapeutic antibody currently employed in the treatment of metastatic stages of breast cancer. Herein, we propose a simple, fast and cost-effective methodology to conjugate trastuzumab with 22-mer 5' thiol-modified oligonucleotides using a bifunctional crosslinker. The conjugates were successfully characterized by MALDI-ToF MS and SDS-PAGE, obviating the need for enzymatic digestion and difficult chromatographic separations. Furthermore, ELISA was performed to ensure that trastuzumab activity is not affected by oligonucleotide conjugation.

Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.

Control of mRNA decay by heat shock-ubiquitin-proteasome pathway.

Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.

Calcium signaling by HBx protein in hepatitis B virus DNA replication.

Hepatitis B virus (HBV) infects more than 300 million people and is a leading cause of liver cancer and disease. The HBV HBx protein is essential for infection; HBx activation of Src is important for HBV DNA replication. In our study, HBx activated cytosolic calcium-dependent proline-rich tyrosine kinase-2 (Pyk2), a Src kinase activator. HBx activation of HBV DNA replication was blocked by inhibiting Pyk2 or calcium signaling mediated by mitochondrial calcium channels, which suggests that HBx targets mitochondrial calcium regulation. Reagents that increased cytosolic calcium substituted for HBx protein in HBV DNA replication. Thus, alteration of cytosolic calcium was a fundamental requirement for HBV replication and was mediated by HBx protein.

Inhibition of Cap-initiation complexes linked to a novel mechanism of eIF4G depletion in acute myocardial ischemia.

Translational control in the rat heart was characterized during acute myocardial ischemia introduced by left coronary artery ligature. Within 10 min of ischemia, eukaryotic (eIF)4E binds to its negative regulator, eIF4E-binding protein-1 (4E-BP1), but the levels of 4E-BP1 are insufficient to disrupt cap-dependent mRNA initiation complexes. However, by 1 h of ischemia, the abundance of the cap-initiation complex protein eIF4G is reduced by relocalization into TIAR protein complexes, triggering 4E-BP1 sequestration of eIF4E and disruption of cap-dependent mRNA initiation complexes. As the heart begins to fail at 6 h, proteolysis of eIF4G is observed, resulting in its depletion and accompanied by limited destruction of 4E-BP1 and eIF4E. eIF4G proteolysis and modest loss of 4E-BP1 are associated with caspase-3 activation and induction of cardiomyocyte apoptotic and necrotic death. Acute heart ischemia therefore downregulates cap-dependent translation through eIF4E sequestration triggered by eIF4G depletion.

The role and fate of rabbit and human transcobalamin II in the plasma transport of vitamin B12 in the rabbit.

Previous studies have shown that plasma transcobalamin II (TCII) facilitates the cellular uptake of [57Co] vitamin B12 (B12) by a variety of tissues, but the lack of an intrinsic label on the protein moiety of the TCII-B12 complex has made it impossible to determine the role and fate of TCII during this process. We have labeled homogensous rabbit and human TCII with 125I-labeled N-succinimidyl-3-(4-hydroxyphenyl) propionate and have performed in vivo experiments in rabbits. When 125I-labeled rabbit TCII-[57Co] B12 and 131I-labeled bovine albumin were simultaneously injected intravenously, we observed that 125Iand 57Co were cleared from plasma at a faster rate (t1/2 = 1 1/2 h) than 131I and that 125I and 57Co were present in excess of 131I in the kidney, liver, spleen, heart, lung, and small intestine 1/2 h after injection. Later, 57Co remained in excess of 131I, but the ratio of 125I to 131I decreased progressively in all of these plasma and were rapidly excreted in the urine. After 1 h following injection, 57Co was present in excess of 125I in the plasma...

Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism.

The 3' noncoding region element (AUUUA)n specifically targets many short-lived mRNAs for degradation. Although the mechanism by which this sequence functions is not yet understood, a potential link between facilitated mRNA turnover and translation has been implied by the stabilization of cellular mRNAs in the presence of protein synthesis inhibitors. We therefore directly investigated the role of translation on mRNA stability. We demonstrate that mRNAs which are poorly translated through the introduction of stable secondary structure in the 5' noncoding region are not efficiently targeted for selective destabilization by the (AUUUA)n element. These results suggest that AUUUA-mediated degradation involves either a 5'-->3' exonuclease or is coupled to ongoing translation of the mRNA. To distinguish between these two possibilities, we inserted the poliovirus internal ribosome entry site, which promotes internal ribosome initiation, downstream of the 5' secondary structure. Translation directed by internal ribosome binding was found to fully restore targeted destabilization of AUUUA-containing mRNAs despite the presence of 5' secondary structure. This study therefore demonstrates that selective degradation mediated by the (AUUUA)n element is coupled to ribosome binding or ongoing translation of the mRNA and does not involve 5'-to-3' exonuclease activity.

Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras.

The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.

Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation.

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.

An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.

Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE.

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.

Role of Ser-53 phosphorylation in the activity of human translation initiation factor eIF-4E in mammalian and yeast cells.

Eukaryotic translation initiation factor eIF-4E is essential for protein synthesis and cell viability. eIF-4E participates in formation of an m7GTP-cap binding protein complex that mediates association of 40S ribosomal subunits with mRNAs, which occurs only when eIF-4E is phosphorylated. Regulation of eIF-4E by phosphorylation was thought to occur on Ser53, although results potentially inconsistent with phosphorylation of this site have been reported. To resolve whether Ser53 is phosphorylated, and if so whether it regulates eIF-4E activity, we directly examined whether Ser53 is a site for phosphorylation of mammalian eIF-4E in human and yeast cells. Wild-type (wt) human eIF-4E protein variants, Ser53-->Asp53 or Ser53-->Ala53, were constructed and analyzed by overproduction in transfected human 293/T-Ag cells, or in Saccharomyces cerevisiae in which the endogenous eIF-4E gene was disrupted. Wt eIF-4E and Ser53 mutants functioned equally well in protein synthesis in both systems, and were phosphorylated to the same extent. Most importantly, the wt and Ser53 mutants of human eIF-4E produced identical tryptic phophopeptide patterns in human cells, and identical but more complicated patterns in yeast. These data demonstrate that Ser53 is not a requisite activating site for phosphorylation of mammalian eIF-4E in human or yeast cells, under conditions in which it participates in protein synthesis.

Familial oculoleptomeningeal amyloidosis. Report of a new family with unusual features.

A family had a dominantly inherited amyloid angiopathy that involved the meninges of the brain and spinal cord, retina, vitreous humor, peripheral nerves, and systemic organs. Clinical features included hemiplegic migraine, periodic obtundation, psychosis, seizures, intracerebral hemorrhage, myelopathy, visual impairment, deafness, and peripheral neuropathy. Pathological findings consisted of amyloid deposition in the leptomeningeal and retinal vessels, in the vitreous humor, and in perivascular tissue throughout the body. Evaluation of the amyloid showed it to be a transthyretin (prealbumin). A brief course of plasmapheresis produced a short-lived decrease concentration in circulating transthyretin.

Quantification of flow in a dynamic phantom using Rb81-Kr81m, and a Nal detector.

Blood flow can be measured by monitoring the count rate of Krypton-81m after its parent, Rubidium-81 (a potassium analogue), has been deposited in the tissue. The steady-state Kr-81m count rate reflects both production by decay of Rb-81 and washout due to blood flow. Its use is theoretically superior to that of Xenon-133 for quantification of blood flow (cc/min per 100 gm) since: (1) multiple flow measurements can be obtained from a single arterial injection, (2) flow-dependent changes in the count rate of Kr-81m provide a steady-state measure of specific flow, and (3) errors due to uptake in fat are eliminated. The count rate of Kr-81m was measured as a function of flow in a dynamic phantom with a NaI crystal, suing both pure cyclotron-produced Rb-81 and commercially available samples with as much as 25% contamination from Rb-82m. The phantom was calibrated by measuring the flow-rate constants with Xe-133. No significant difference was found between the flow-rate constant measured with three pure samples. The ratio of the zero-flow Kr-81m count rate to the rate observed in the presence of flow showed excellent correlation with calibrated flow over a range of rate constant from 0 to 0.02 sec (-1). This study suggests that regional specific flow can be measured in vivo with currently available Nal detecting systems after the intra-arterial injection of Rb-81.

Proton irradiation of choroidal melanomas. Preliminary results.

Choroidal malignant melanomas in nine patients were treated with proton beam irradiation at the Harvard Cyclotron Laboratory, Cambridge, Mass. Each patient received five proton beam treatments in eight to ten days, totalling 4,730 to 8,570 rads at the tumor. No complications occurred during the treatment or follow-up period, which, at the time of this writing, ranges from one to 24 months, with an average of 12 months. No further growth of the tumor has been observed in any patient. Different signs of tumor regression have been noted. Resolution of the serous retinal detachments that accompanied some tumors is the earliest finding. Pigment changes over the surface of the tumor and adjacent pigment epithelium is a usual initial tumor response. Fluorescein angiography initially showed decreased leakage of dye; later, destruction of the tumor's vasculature and elimination of fluorescein leakage became evident. Only large choroidal vessels remained patient. Ultrasonography revealed decreased height of the tumors postirradiation, and the radioactive phosphorus (32P) uptake test, repeated in one patient, turned negative on postirradiation measurements.

Immunohistochemical detection of hepatitis C antigen by monoclonal antibody TORDJI-22 compared with PCR viral detection.

We sought to determine the sensitivity and specificity of immunohistochemistry using the TORDJI-22 MoAb (BioGenex, San Ramon, Calif), which is specific for the C-100 protein of the hepatitis C virus, compared with reverse transcriptase-polymerase chain reaction (RT-PCR) of tissue for viral RNA. RT-PCR had been performed on 52 fixed tissue specimens. Immunohistochemistry was performed using prediluted antibody with the alkaline phosphatase/fast red (BioGenex) technique. Predigestion with Protease XXIV (BioGenex) and other procedures followed the manufacturer's protocols. Positive immunohistochemistry was narrowly defined as tightly clumped, perinuclear red granules in hepatocytes. Of the specimens, 28 were positive by RT-PCR. With RT-PCR as the standard of comparison, immunohistochemistry yielded a sensitivity of 70% and specificity of 84%. Positive cells, when present, were usually very rare. With stringent criteria, immunohistochemistry with the TORDJI-22 monoclonal antibody is a very specific, fairly sensitive diagnostic test for hepatitis C virus in fixed liver tissues.

Persistence of virus-specific IgM and clinical recovery after Japanese encephalitis.

We have searched for evidence of a chronic Japanese encephalitis virus (JEV) infection in six Thai patients convalescing from acute Japanese encephalitis (JE) in whom JEV-specific IgM antibody was last detected 116 to 350 days after their acute illness. These six patients were compared with 94 other JE patients matched for age, sex and serological response and in whom JEV-specific IgM was either short-lived (less than 90 days) or not tested. All patients were evaluated for the presence or absence of seven abnormal neurological signs over a 1- to 2-year period. During the first 30 days of illness the mean numbers (+/- S.E.M.) of abnormal signs per patients for the IgM and control groups were 3.8 +/- 0.3 and 2.3 +/- 0.1, respectively (P less than 0.01). After 1 year the six IgM patients still had significantly more abnormal neurological signs than controls (1.3 +/- 0.3 and 0.6 +/- 0.1, respectively [P less than 0.01]). By 2 years, the IgM group showed no neurological impairment; examination of cerebrospinal fluids revealed no evidence of subclinical viral infections. The recovery of the six IgM patients between 1 and 2 years after their relatively severe acute illness suggests that IgM antibody persistence was related to acute virulence rather than chronicity of the JEV infection.

A modality-specific somatosensory area within the insula of the rhesus monkey.

Response properties of neurons in the monkey's granular insula (Ig) were examined with somatic, auditory, visual, and gustatory stimuli. Results indicate that a major portion of Ig is a somatic processing area exclusively, with units that have large and often bilateral receptive fields, consistent with the view that this area serves as a higher-order, modality-specific link in the somatosensory-limbic pathway.