Mitogenic factors present in serum but not in plasma.

In culture medium containing heparinized, heat-inactivated, chicken plasma, normal chicken heart mesenchymal cells do not proliferate but their Rous sarcoma virus-infected counterparts proliferate maximally. In medium containing serum derived from chicken whole blood or plasma, on the other hand, normal chicken heart mesenchymal cells proliferate actively, at similar overall rates and to similar extents. The rate and extent of normal cell proliferation are decreased by a factor of approximately 1/2 with whole blood-derived serum that is heparinized and inactivated; proliferation ceases in plasma-derived serum that is heparinized and inactivated. Heparinization and inactivation of serum does not affect the proliferation of Rous sarcoma virus-infected cells, indicating that this combined treatment eliminates a mitogenic (regulatory) rather than a supportive (nutrient) factor(s) for cell replication. We hypothesize that mitogen(s) is released from plasma protein precursors when plasma clots in the presence of formed elements of the blood or when plasma-derived serum is exposed to cultured cells; heparinization and inactivation, within the framework of this hypothesis, would render nonfunctional the plasma protein precursor(s) from which the mitogen(s) is generated. Alternatively, our data are consistent with the release of two mitogens during blood clotting, one from plasma protein precursors and the other from formed elements of the blood. We also have studied the proliferative behavior of Swiss and BALB/c 3T3 cells in whole blood-derived and plasma-derived human serum. Our studies suggest that the platelet-derived growth factor has an artifactual supportive (nutrient) role, rather than an authentic mitogenic role, in cell replication.     

Non-invasive sample collection for respiratory virus testing by multiplex PCR

Background

Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.

Objective

Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR.

Study design

Children aged 1 month–17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard.

Results

285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥ 1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods.

Conclusions

Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.     

Immunohistochemical analysis of receptor tyrosine kinase signal transduction activity in chordoma

AIMS: Currently, there are no effective chemotherapeutic protocols for chordoma. Reports of receptor tyrosine kinase (RTK) expression in chordoma suggest that these tumours may respond to kinase inhibitor therapy. However, RTK signalling activity has not been extensively investigated in chordoma. METHODS: A tissue microarray containing 21 cases of chordoma was analysed for expression of a number of proteins involved in signal transduction from RTKs by immunohistochemistry. RESULTS: Platelet-derived growth factor receptor-beta, epidermal growth factor receptor (EGFR), KIT and HER2 were detected in 100%, 67%, 33% and 0% of cases, respectively. Platelet-derived growth factor receptor-beta staining was of moderate-to-strong intensity in 20 of 21 cases. In contrast, KIT immunoreactivity was weak and focal in each of the seven positive cases. Total EGFR staining was variable; weak staining for phosphorylated EGFR was detected in nine cases. Phosphorylated isoforms of p44/42 mitogen-activated protein kinase, Akt and STAT3, indicative of tyrosine kinase activity, were detected in 86%, 76% and 67% of cases, respectively. CONCLUSIONS: Chordomas commonly express RTKs and activated signal transduction molecules. Although there were no statistically significant correlations between the expression of any of the markers studied and disease-free survival or tumour location, the results nonetheless indicate that chordomas may respond to RTK inhibitors or modulators of other downstream signalling molecules.     

Proteomic Profiling of Paraffin-Embedded Samples Identifies Metaplasia-Specific and Early-Stage Gastric Cancer Biomarkers

Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis.Despite the general advances in endoscopic screening and therapies, gastric cancer 5-year survival rates remain extremely poor,¹ representing the second leading cause of cancer-related death worldwide. The major proximate cause of gastric cancer is chronic Helicobacter pylori infection, which leads to a chronic inflammatory response and subsequent oxyntic atrophy (loss of acid-secreting parietal cells). In the fundus and corpus of the atrophic stomach, two types of metaplasia have been described: intestinal metaplasia (IM), characterized by the presence of cells with intestinal and goblet cell morphologic features, and spasmolytic polypeptide–expressing metaplasia (SPEM), which shows morphologic characteristics of the deep antral glands and expresses trefoil factor 2 (TFF2), originally designated spasmolytic polypeptide.^2,3Both types of metaplasia are associated with intestinal-type gastric cancer^4,5 and are considered neoplastic precursors, although the mechanisms driving the progression from metaplasia to neoplasia remain unclear. Recent studies in mice have found that SPEM originates from the transdifferentiation of mature chief cells.⁶ Other studies in Mongolian gerbils indicate that after H. pylori–induced parietal cell loss, SPEM is the first metaplastic lesion to evolve, whereas IM develops in the setting of preexisting SPEM.^7,8 Recent pathologic examinations in humans have suggested that a similar relationship between SPEM and IM may exist in humans.^9,10Molecular profiling studies have identified a variety of potentially useful markers for gastric cancer.^11–14 However, owing to the high heterogeneity of gastric tumors, no definitive markers have been established. High levels of REG4 were detected in patients with gastric cancer with metastasis, and its expression was correlated with worse prognosis.^14,15 Other studies have noted that REG4 contributes to the resistance of gastric cancer to fluorouracil-based chemotherapy; in addition, patients with gastric cancer showed increased levels of serum REG4. However, despite a specificity of 99%, the diagnostic sensitivity was only 36%.^14,16 OLFM4 expression has been associated with intestinal-type gastric cancer. However, different proportions of OLFM4-positive tumors were observed in two independent studies (65%¹⁷ in contrast to 32%¹¹), and its prognostic value is still not clear.^11,18 Inclusion of the less heterogenous metaplastic lesions in the molecular profiling studies could allow a better understanding of the molecular alterations during gastric carcinogenesis and could lead to the development of early-stage gastric cancer biomarkers. Indeed, in a previous study, mRNA expression profiling of IM and SPEM identified several metaplasia and gastric cancer markers, including CDH17 and MUC13, as useful prognostic markers for stage I gastric cancer.¹¹ The combined loss of four metaplasia markers (CDH17, REG4, MUC13, and LGALS4) is an independent indicator of survival in undifferentiated or stage II/III gastric cancer.¹⁹Formalin-fixed, paraffin-embedded (FFPE) tissue samples are abundantly available in pathology archives, representing a valuable resource for biomarker discovery. Recent developments have facilitated the analysis of proteomic profiles from paraffin-embedded tissues with yields in the range of 90% of the peptides isolated from frozen tissue, showing good equivalence between the protein profiles from frozen and FFPE samples.^20,21 This innovation means that proteomic analysis can be performed on paraffin-embedded tissue samples with defined pathologic and histologic characteristics, allowing more specific analyses in better-characterized samples.In the present studies, we performed proteomic profiling using macrodissected FFPE samples from intestinal-type gastric cancer, stomach metaplasia, and normal mucosa. These studies identified a variety of proteins that are up-regulated in metaplasia and cancer. We identified lactotransferrin (LTF) as a novel specific marker for SPEM and deleted in malignant brain tumor 1 (DMBT1) as a marker for IM. In addition, we found that expression of either LTF or DMBT1 influences the survival of patients with gastric cancer.