A case with glucagonoma syndrome--heterogeneity of glucagon and insulin

The heterogeneity of glucagon and insulin in plasma and tissue extracts from a 57-year-old female with glucagonoma syndrome with surgically and autopsy verified islet-cell tumors was studied by Bio-Gel P-10 filtration. The preoperative plasma immunoreactive glucagon (IRG) level was 20.2 ng/ml, and plasma glucagon-like immunoreactivity(GLI) 25.8 ng/ml. The column chromatography of the preoperative plasma revealed three or four IRG components and four GLI components. Among these, peak II, the large glucagon immunoreactivity (LGI) peak, considered a candidate for proglucagon, was prominent, along with peak III. The resected metastatic liver tumor contained an enormous amount of IRG and an appreciable amount of immunoreactive insulin (IRI), indicating that the elevated plasma IRG was mainly of tumor origin. The IRG pattern of the tumor tissue extract revealed a small quantity of IRG in peaks I and II, and a large amount in peak III; control pancreatic tissue extract manifested a similar elution pattern. The IRI elution pattern of the tumor tissue extract revealed two major IRI peaks which migrated close to the elution volume of cytochrome C and insulin, respectively. This is a quite different pattern from the control pancreatic tissue extract in which the RI peak was localized in the elution volume of the insulin. We conclude that the present metastatic liver tumor produced not only enormous amounts of glucagon but heterogeneous peptides which contained immunological insulin determinants within their.     

Treatment with danazol and plasma glucagon concentration.

The association between treatment with danazol and hyperglucagonaemia was studied. Plasma glucagon concentrations were measured during an oral glucose tolerance test in seven women taking danazol and six healthy controls not taking danazol. Results showed that treatment with danazol is associated with severe hyperglucagonaemia, and in three patients glucagon concentrations reached the range suggestive of glucagonoma. It is important to recognise that this increasingly used drug may cause severe hyperglucagonaemia to prevent patients treated with danazol undergoing unnecessary investigations to localise glucagonoma.     

Characterizing mutational heterogeneity in a glioblastoma patient with double recurrence

Human cancers are driven by the acquisition of somatic mutations. Separating the driving mutations from those that are random consequences of general genomic instability remains a challenge. New sequencing technology makes it possible to detect mutations that are present in only a minority of cells in a heterogeneous tumor population. We sought to leverage the power of ultra-deep sequencing to study various levels of tumor heterogeneity in the serial recurrences of a single glioblastoma multiforme patient. Our goal was to gain insight into the temporal succession of DNA base-level lesions by querying intra- and inter-tumoral cell populations in the same patient over time. We performed targeted "next-generation" sequencing on seven samples from the same patient: two foci within the primary tumor, two foci within an initial recurrence, two foci within a second recurrence, and normal blood. Our study reveals multiple levels of mutational heterogeneity. We found variable frequencies of specific EGFR, PIK3CA, PTEN, and TP53 base substitutions within individual tumor regions and across distinct regions within the same tumor. In addition, specific mutations emerge and disappear along the temporal spectrum from tumor at the time of diagnosis to second recurrence, demonstrating evolution during tumor progression. Our results shed light on the spatial and temporal complexity of brain tumors. As sequencing costs continue to decline and deep sequencing technology eventually moves into the clinic, this approach may provide guidance for treatment choices as we embark on the path to personalized cancer medicine.     

Suppression of plasma prolactin in a patient with a pinealocytoma

Plasma growth hormone (HGH), prolactin (PRL), luteinizing hormone (LH), thyroid stimulating hormone (TSH), cortisol and melatonin were determined during a 24 h period in a pubertal boy with a pinealocytoma. All hormone concentrations were normal with respect to age and time of day, with the exception of PRL which was undetectable. After subtotal removal of the tumor, basal PRL was still undetectable, but could be stimulated moderately by insulin-induced hypoglycaemia or TRH.     

Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity

We describe a bioinformatic tool, Tumor Aberration Prediction Suite (TAPS), for the identification of allele-specific copy numbers in tumor samples using data from Affymetrix SNP arrays. It includes detailed visualization of genomic segment characteristics and iterative pattern recognition for copy number identification, and does not require patient-matched normal samples. TAPS can be used to identify chromosomal aberrations with high sensitivity even when the proportion of tumor cells is as low as 30%. Analysis of cancer samples indicates that TAPS is well suited to investigate samples with aneuploidy and tumor heterogeneity, which is commonly found in many types of solid tumors.     

Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity

We describe a bioinformatic tool, Tumor Aberration Prediction Suite (TAPS), for the identification of allele-specific copy numbers in tumor samples using data from Affymetrix SNP arrays. It includes detailed visualization of genomic segment characteristics and iterative pattern recognition for copy number identification, and does not require patient-matched normal samples. TAPS can be used to identify chromosomal aberrations with high sensitivity even when the proportion of tumor cells is as low as 30%. Analysis of cancer samples indicates that TAPS is well suited to investigate samples with aneuploidy and tumor heterogeneity, which is commonly found in many types of solid tumors.     

Secretory granule-mediated co-secretion of L-glutamate and glucagon triggers glutamatergic signal transmission in islets of Langerhans

L-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans. However, critical issues, i.e. where, when and how L-glutamate appears, and what happens upon stimulation of glutamate receptors in the islets, remain unresolved. Vesicular glutamate transporter 2 (VGLUT2), an isoform of the vesicular glutamate transporter essential for neuronal storage of L-glutamate, is expressed in alpha cells (Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001) J. Biol. Chem. 276, 43400-43406). Here we show that VGLUT2 is specifically localized in glucagon-containing secretory granules but not in synaptic-like microvesicles in alpha TC6 cells, clonal alpha cells, and islet alpha cells. VGLUT1, another VGLUT isoform, is also expressed and localized in secretory granules in alpha cells. Low glucose conditions triggered co-secretion of stoichiometric amounts of L-glutamate and glucagon from alpha TC6 cells and isolated islets, which is dependent on temperature and Ca(2+) and inhibited by phentolamine. Similar co-secretion of L-glutamate and glucagon from islets was observed upon stimulation of beta-adrenergic receptors with isoproterenol. Under low glucose conditions, stimulation of glutamate receptors facilitates secretion of gamma-aminobutyric acid from MIN6 m9, clonal beta cells, and isolated islets. These results indicate that co-secretion of L-glutamate and glucagon from alpha cells under low glucose conditions triggers GABA secretion from beta cells and defines the mode of action of L-glutamate as a regulatory molecule for the endocrine function. To our knowledge, this is the first example of secretory granule-mediated glutamatergic signal transmission.     

Study of structural heterogeneity of polymerized hemoglobin using biochemical and immunochemical methods

The structural heterogeneity of crosslinked pyridoxalated hemoglobin (polyHb-PLP) have been investigated by the methods of gel-filtration, isoelectric focusing and immunoelectrophoresis. It was established that the heterogeneity of poly-Hb-PLP is mainly depicted by polydispersion of molecular weight and less by the changes in the isoelectric properties. The qualitative difference has been shown between the immunochemical properties of the native hemoglobin and poly-Hb-PLP, which gives the suggestion of the formation of new antigenic determinants as a result of chemical modification and of more expressed antigenicity of modified hemoglobin compared with native protein. Poly-Hb-PLP has wide antigenic spectrum that corresponds to its structural heterogeneity. The data obtained suggest that the decrease in the antigenic activity of poly-Hb-PLP can be achieved by the lowering of the part of high molecular weight components.     

Zinc-induced reduction in melphalan cytotoxicity: heterogeneity of response of cloned human tumor cells

Previous studies with cultured human normal fibroblasts indicated that pretreatment of the cells with zinc for 12 h prior to exposure to the alkylating agent melphalan increased survival by seven- to ninefold over survival values obtained in cultures treated with drug only. Comparable pretreatment of cells derived from a variety of human tumors resulted in an increase in survival of 1.7-fold or less. To determine whether the limited responsiveness to zinc represented a general property of tumor cells (which would be characterized by a lack of highly zinc-responsive subpopulations contained within the parental tumor populations), a series of clones was prepared from the A101D human melanoma line and the A549 human alveolar cell carcinoma line. Cells from each clone were then challenged with melphalan with and without zinc pretreatment. Twenty-five percent of the tumor clones exhibited increased resistance to melphalan following pretreatment with zinc (range of 2.1- to 5.2-fold increase in survival), indicating that the parental tumor lines were highly heterogenous in regard to inducibility to a state of reduced sensitivity to melphalan. There was no evidence of a relationship between zinc-induced reductions in toxicity and induced elevations in total intracellular glutathione content, indicating that the primary effect of zinc is not directed toward elevating intracellular levels of glutathione.     

The heterogeneity of the plasma membrane H+-ATPase response to auxin

The sensitivity of the plasma membrane H⁺-ATPase in tobacco was investigated in vitro, both at the proton translocation level and the ATPase level, according to plant development and leaf location. Both activities are stimulated by auxin in all leaves, whatever the plant age and the leaf age. However, the sensitivity to auxin was heterogeneous with respect to plant development and leaf location. In parallel experiments using the same plasma membrane samples, polypepides patterns were investigated by two-dimensional gel electrophoresis and image analysis was used to quantify the relative abundance of 110 peptides. Systematic analysis of the two kinds of data identified 8 polypeptides, the abundance of which changed in a consistent way with the sensitivity, whatever the plant developmental state and leaf location. These unknown polypeptides are proposed as potential markers of the membrane response to auxin.     

Size heterogeneity of immunoreactive glucagon during bile-duct obstruction in the rabbit

The influence of bile-duct obstruction upon basal plasma glucagon levels and the relationship between glucagon-like substance in bile and postobstructive plasma glucagon were examined in the rabbit. Immunoreactive glucagon (IRG) was measured with antiserum 30 K (Unger). Bile-duct obstruction was followed by a prompt rise within 60 minutes in plasma IRG which was four times the basal value, but had little influence on plasma immunoreactive insulin and blood sugar. The biliary IRG and the elevated plasma IRG during bile-duct obstruction were filtered with a Bio-Gel P-10 column. Most of the postobstructive plasma IRG appeared in the void volume area (plasma large IRG), while almost all of the biliary IRG was recovered in the position equivalent to approximately 2000 daltons (biliary IRG 2000). Both IRGs of different molecular sizes revealed similar dilution curve in radioimmunoassay to that with porcine glucagon. After incubation of bile with preobstructive plasma, the IRG elution profile of the mixture contained an increased amount of large molecular size IRG similar to that of postobstructive plasma in regard to 30K specificity and elution position. The disappearance of IRG in the void volume area was observed when the bile-plasma mixture or the postobstructive plasma was filtered with acidic buffer. These results suggest that plasma large IRG contributing to hyperglucagonemia during bile-duct obstruction may be derived from biliary IRG 2000.     

Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma

Background

Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets.

Patients and methods

This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS).

Results

Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR?+?PR?+?SD) more often showed SSTRs than patients with disease progression (p?=?0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6?months; p?=?0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p?=?0.013). The induction of SSTRs was associated with gender (female vs. male; p?=?0.014) and disease stage (M1a/b vs. M1c; p?=?0.010), but not with patient age, HLA type, performance status, or vaccination regimen.

Conclusion

Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma.