Facts and fiction: premalignant lesions of lung tissues.

Lung cancer is now the leading cause of death from cancer in Australia. Most patients are diagnosed with late-stage disease. Although diagnosis at pre-invasive stages could theoretically improve outcomes, mooted precursor lesions are often asymptomatic and often undetectable by non-invasive investigations. Nonetheless, they merit study to identify early and essential molecular steps involved in lung carcinoma pathogenesis, with the aim of developing therapies targeted against one or more such steps. Some lung cancers appear to develop via a series of progressive morphological changes with correlating molecular alterations, but others seem to arise in histologically normal epithelium, and these differences may reflect anatomically and functionally distinct epithelial compartments of the respiratory tract. Pre-invasive precursor lesions recognised by the World Health Organization (WHO) include squamous metaplasia with dysplasia and carcinoma in situ, atypical adenomatous hyperplasia, and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. Other lesions that likely represent pre-invasive lesions, but which are not currently WHO-listed, include human papillomavirus (HPV)-related respiratory papillomatosis and mesothelioma in situ. No single cancer stem cell marker has been identified. Field cancerisation plays an important role in lung cancer development, and includes the spread of pre-invasive clones along the respiratory epithelium or the occurrence of multiple separate foci of pre-invasive abnormalities such as squamous dysplasia and carcinoma in situ.In addition to well-characterised step-wise progression in squamous cell carcinomas and some adenocarcinomas, alternative pathways exist, and are currently being investigated. In addition, molecular techniques, including miRNA screening on blood samples or cytology samples--such as sputum samples--may become clinically relevant and more accurate in predicting lung cancer progression.

1-Methylguanosine in place of Y base at position 37 in phenylalanine tRNA is responsible for its shiftiness in retroviral ribosomal frameshifting.

Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNA(Phe) contains 1-methylguanosine (m(1)G) in place of Y and that the m(1)G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m(1)G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2'-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3' side of the anticodon render tRNA(Phe) shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed.

The ribonucleotide reductase R2 gene is a non-transcribed target of c-Myc-induced genomic instability.

The c-Myc oncoprotein is highly expressed in malignant cells of many cell types, but the mechanism by which it contributes to the transformation process is not fully understood. Here, we show for the first time that constitutive or activated overexpression of the c-myc gene in cultured mouse B lymphocytes is followed by chromosomal and extrachromosomal amplification as well as rearrangement of the ribonucleotide reductase R2 gene locus. Electron micrographs and fluorescent in situ hybridization (FISH) demonstrate the c-Myc-dependent generation of extrachromosomal elements, some of which contain R2 sequences. However, unlike other genes that have been shown to be targets of c-Myc-dependent genomic instability, amplification of the R2 gene is not associated with alterations in R2 mRNA or protein expression. These data suggest that c-Myc-dependent genomic instability involves a greater number of genes than previously anticipated, but not all of the genes that are amplified in this system are transcriptionally upregulated.

Chromosomal and extrachromosomal instability of the cyclin D2 gene is induced by Myc overexpression.

We examined the expression of cyclins D1, D2, D3, and E in mouse B-lymphocytic tumors. Cyclin D2 mRNA was consistently elevated in plasmacytomas, which characteristically contain Myc-activating chromosome translocations and constitutive c-Myc mRNA and protein expression. We examined the nature of cyclin D2 overexpression in plasmacytomas and other tumors. Human and mouse tumor cell lines that exhibited c-Myc dysregulation displayed instability of the cyclin D2 gene, detected by Southern blot, fluorescent in situ hybridization (FISH), and in extrachromosomal preparations (Hirt extracts). Cyclin D2 instability was not seen in cells with low levels of c-Myc protein. To unequivocally demonstrate a role of c-Myc in the instability of the cyclin D2 gene, a Myc-estrogen receptor chimera was activated in two mouse cell lines. After 3 to 4 days of Myc-ER activation, instability at the cyclin D2 locus was seen in the form of extrachromosomal elements, determined by FISH of metaphase and interphase nuclei and of purified extrachromosomal elements. At the same time points, Northern and Western blot analyses detected increased cyclin D2 mRNA and protein levels. These data suggest that Myc-induced genomic instability may contribute to neoplasia by increasing the levels of a cell cycle-regulating protein, cyclin D2, via intrachromosomal amplification of its gene or generation of extrachromosomal copies.

Reactive mesothelial hyperplasia vs mesothelioma, including mesothelioma in situ: a brief review.

In biopsy tissue, discrimination between reactive mesothelial hyperplasia and epithelial mesothelioma can pose a major problem for the surgical pathologist. Confidence in the diagnosis is often proportional to the amount of tissue available for study and depends largely on findings of invasion and the extent and cytologic atypia of the lesion, because there is no marker specific for the mesothelium and that discriminates consistently among normal, hyperplastic, and neoplastic mesothelial tissue. Therefore, mesothelioma in situ is diagnosable only when invasive epithelial mesothelioma is demonstrable in the same specimen, in a follow-up biopsy specimen, or at autopsy. Comparison of 22 cases of mesothelioma in situ that fulfill these requirements for diagnosis with 141 invasive mesotheliomas and 78 reactive mesothelioses indicates that strong linear membrane-related labeling for epithelial membrane antigen and silver-labeled nucleolar organizer region-positive material that occupies 0.6677 microm2 or more of the nucleus in an atypical in situ mesothelial lesion of the pleura are found consistently in neoplastic mesothelial cells. Although these findings may engender suspicion of mesothelioma in situ in high-risk persons, the criteria for diagnosis of pure mesothelial lesions of this type are still under study. Mesothelioma in situ should be considered proved only when unequivocal invasion is identified in a different area of the pleura or at a different time; a diagnosis of pure mesothelioma in situ should not be made in patients not exposed to asbestos.

Activation of bcl-2 suppressible 40 and 44 kDa p38-like kinases during apoptosis of early and late B lymphocytic cell lines.

Activation of several different kinases characterizes the induction of apoptosis. Abelson virus transformed pre-B lymphocytes undergo apoptosis within 24 h of serum deprivation, PKA activation or gamma-irradiation, and the activity of two kinases of ca. 40 and 44 kDa is specifically induced during this apoptotic process. Bcl-2 expression prevents both apoptosis and the induction of these kinases. Immunologic and substrate similarities indicate that these kinases are related to the p38 family of MAP kinases. More mature cells of the B lymphocytic lineage, plasmacytomas, also exhibit induction of these kinases when apoptosis is induced by withdrawal of serum or IL-6. Treatment of the pre-B cells with ICE protease inhibitors when apoptotic stimuli are delivered prevents induction of the kinase activity, and partially inhibits apoptosis. These findings indicate that the induction of these 40 and 44 kDa p38 related kinases is a common feature of apoptosis in mouse B lymphocytic cells and may represent a step downstream of ICE proteases in the signal cascade that leads to programmed cell death.

The use of epithelial membrane antigen and silver-stained nucleolar organizer regions testing in the differential diagnosis of mesothelioma from benign reactive mesothelioses.

BACKGROUND: The accurate diagnosis of pleural lesions obtained from small closed biopsy is difficult. As yet there is no single reliable test to distinguish between malignant and benign mesothelial tissue. METHODS: Immunostaining of epithelial membrane antigen (EMA) and the quantitation of silver stained nucleolar organizer regions (AgNORs) each were applied to benign and malignant histologic sections of pleural and peritoneal biopsies. The usefulness of these stains was tested both individually and in combination in the diagnosis of epithelial malignant mesothelioma. RESULTS: One hundred and three of the 141 malignant lesions (73%) were immunoreactive for EMA but only 3 of the 73 benign lesions (4%) reacted equivocally, and none positively. The average count of AgNORs/cell in malignant lesions (n = 80) was elevated compared with benign cases (n = 26), but a significant overlap was exhibited in the AgNOR count and this form of analysis was considered to be of little value in distinguishing benign from malignant mesothelial processes. Much less overlap was observed when the average AgNOR area was measured. By using the maximum benign AgNOR area of 0.6677 microm2 as the upper threshold, 51 cases (63.8%) were identified as malignant; the test demonstrated 100% specificity and 63.8% sensitivity. By combining the EMA and AgNOR results, 76 of 80 of the malignant mesothelioma cases (95%) tested positive for at least 1 of the tests with no false-positive results identified. CONCLUSIONS: This study confirms the usefulness of EMA in diagnosing malignant and benign mesothelial lesions, and demonstrates the enhanced diagnostic value of combining EMA immunoreaction with the average area of AgNOR per cell, thereby increasing sensitivity in the diagnosis of epithelial malignant mesothelioma.

Crystalloidal paraprotein deposits in the cornea: an ultrastructural study of two new cases with tubular crystalloids that contain IgG kappa light chains and IgG gamma heavy chains.

The fine structure and immunoprotein content of the crystalloids are described in two cases of paraproteinemic crystalloidal keretopathy, both of which had clinical features thought by the referring ophthalmologists to be those of atypical lattice-type corneal dystrophy (presumably because of lattice-like lines). Most keratocytes in one case were surrounded by a mantle of densely packed tubular crystalloids. Individual tubules were annular in cross section with mean dimensions as follows: overall diameter, 29.32 nm (SD 1.26); internal diameter (core), 8.53 nm (SD 1.12); wall thickness, 10.39 nm (SD 0.85) (n = 10). Crystalloids were extracellular and found only in the corneal stroma, with none in Bowman's layer or Descemet's membrane. In the second case, the tubules had a similar distribution but formed geometric arrays with no clear relationship to, or envelopment of the keratocytes. The tubules were thin-walled, with mean dimensions as follows: overall diameter, 26.12 nm (SD 1.12); internal diameter (core), 15.46 nm (SD 1.12); wall thickness, 5.33 nm (SD 0) (n = 10). In both cases the tubules were kappa-light chain- and gamma-chain-positive. Laboratory investigations revealed the presence of two IgM-kappa paraproteins and an IgG-kappa paraprotein in the serum of the first patient. The second patient had an IgG-kappa paraproteinemia and bone marrow changes consistent with low-grade non-Hodgkin's lymphoma. These cases emphasize and extend the morphological range of corneal IgG crystalloids; the second case also demonstrates that corneal IgG crystalloids may be an early indicator of un underlying immunoproliferative disease.

Asbestos, asbestosis, pleural plaques and lung cancer.

Inhalation of asbestos fibers increases the risk of bronchial carcinoma. It has been claimed that asbestosis is a necessary prerequisite for the malignancy, but epidemiologic studies usually do not have enough statistical strength to prove that asbestos-exposed patients without asbestosis are without risk. Several recent studies do actually indicate that there is a risk for such patients. In addition, case-referent studies of patients with lung cancer show an attributable risk for asbestos of 6% to 23%, which is much higher than the actual occurrence of asbestosis among these patients. Thus there is an increasing body of evidence that, at low exposure levels, asbestos produces a slight increase in the relative risk of lung cancer even in the absence of asbestosis. Consequently, all exposure to asbestos must be minimized.

Epithelioid sarcoma of the forearm.

A case of epithelioid sarcoma of the forearm is described. The radiological and pathological features, natural history of the tumour and its treatment are reviewed.

Overexpression of v-abl uniquely cooperates with c-myc dysregulation in induction of plasma cell tumors, bypassing the need for T-lymphocytic help and overcoming T-lymphocytic interference.

We have investigated the effects of T lymphocytes on induction of mouse plasma cell tumors. We show that ABL-MYC, a plasmacytomagenic retrovirus that constitutively expresses v-abl and c-myc, is able to induce plasmacytomas in 100% of athymic BALB/c mice, with or without intraperitoneal pristane pretreatment. Other induction regimens are ineffective under these conditions, indicating that the combination of v-abl and c-myc oncogenes is uniquely able to transform plasma cells in mice that are deficient in T lymphocytes. Furthermore, in the absence of pristane, ABL-MYC-infected athymic congenics developed plasmacytomas in half the time required for euthymic BALB/c mice, suggesting that T lymphocytes can have a negative effect and can retard, but not totally inhibit, the outgrowth of plasmacytomas. This phenomenon could not be appreciated in other regimens of plasmacytoma induction, because only ABL-MYC is sufficient to induce plasmacytomas in athymic mice or in euthymic mice in the absence of pristane pretreatment.

Protein kinase C-zeta reverts v-raf transformation of NIH-3T3 cells.

We have identified protein kinase C-zeta (PKC-zeta) as a novel suppressor of neoplastic transformation caused by the v-raf oncogene. PKC-zeta overexpression drastically retards proliferation, abolishes anchorage-independent growth, and reverts the morphological transformation of v-raf-transformed NIH-3T3 cells. The molecular basis for this effect appears to be a specific induction of junB and egr-1 expression, triggered synergistically by PKC-zeta via a Raf/Mek/MAPK-independent mechanism and v-raf. junB-promoter/CAT assays revealed that PKC-zeta directly targets the junB promoter. The induction of junB and egr-1 is linked to the v-raf transformation-suppressing effect of PKC-zeta as constitutive expression of junB and egr-1 but not of c-jun also abolishes anchorage-independent growth of v-raf-transformed NIH-3T3 cells. Moreover, junB overexpression leads to a retardation of proliferation in these cells. PKC-zeta interferes with the serum inducibility of an AP-1 reporter plasmid in v-raf-transformed NIH-3T3 cells, indicating that PKC-zeta antagonizes transformation and proliferation by down-modulating AP-1 function via induction of junB. In summary, our data suggest that PKC-zeta counteracts v-raf transformation by modulating the expression of the transcription factors junB and egr-1.

MobyEd: a mobile educator unit.

A mobile educator unit is a cost-effective teaching tool that can be easily implemented in an acute hospital setting to assist in teaching patients, visitors, and personnel about health and wellness. The educator unit cannot replace the face-to-face interactions between professional and patient or visitor, but it can supplement and make information more readily available than a stationary display.

Loss of heterozygosity in asbestos-induced mutations in a human mesothelioma cell line.

The relationship between occupational or environmental exposure to asbestos and the development of mesothelioma, typically after prolonged latency, has been accepted as one of cause and effect. Most studies have concluded that asbestos is not mutagenic to mammalian cells in vitro. We have studied the potential of crocidolite asbestos to induce mutations in a stable mesothelioma cell line, using a mutation assay that measures mutation at the autosomal HLA-A locus and permits clonal growth of mutant cells. The mesothelioma cell line chosen is more akin to the in vivo target cells of asbestos than human peripheral blood lymphocytes used in previous studies. Exposure of mesothelioma cells in culture to both 200 micrograms/ml and 50 micrograms/ml crocidolite for 72 hr did not result in a statistically significant difference in the mutation frequency (MF) in the HLA-A assay when compared to the spontaneous MF in these cells. Mutations in the mesothelioma cells were classified according to their molecular basis. Notwithstanding the lack of statistically significant change in overall MF, molecular analysis of mutants obtained following exposure of mesothelioma cells to crocidolite demonstrated a statistically significant increase in the class of mutations arising from loss of heterozygosity (LOH) events involving the selection locus (HLA-A) and more distal loci. Mutations following exposure to 200 micrograms/ml and 50 micrograms/ml crocidolite showed a greater frequency of LOH than did spontaneous mutants (P < 0.01 and P < 0.001, respectively). These results correlate with those obtained in an earlier study using lymphocytes. The mesothelioma cell-based assay may be useful in detecting the mutagenicity of other asbestiform fibers and man-made fibers.

Asbestos and erionite fibres can induce mutations in human lymphocytes that result in loss of heterozygosity.

Although asbestos and erionite are proven human carcinogens, most studies have concluded that these fibres are not mutagenic to mammalian cells in vitro. We have studied the potential of these fibres and chrysotile fibres to induce mutations in human peripheral lymphocytes, using a mutation assay that measures mutation at the autosomal HLA-A locus. Exposure of lymphocytes in culture to 400 micrograms/ml of crocidolite or erionite for 72 hr did not result in a statistically significant increase in the mutation frequency (MF) in the HLA-A assay, although a trend towards increased MF was observed. Exposure to 400 micrograms/ml chrysotile resulted in no increase in MF; however a significant increase was observed at 50 micrograms/ml. Mutations in somatic cells can be classified according to their molecular basis. Molecular analysis of mutants obtained following exposure of lymphocytes to crocidolite and erionite demonstrated a statistically significant increase in the class of mutations arising from loss-of-heterozygosity (LOH) events involving the selection locus (HLA-A) and more distal loci. Mutations following exposure to crocidolite and erionite showed a greater frequency of LOH than did spontaneous mutants (p < 0.02 and p < 0.005 respectively). Mutants following exposure to chrysotile did not display a significant difference in LOH when compared with spontaneous mutants. Thus, although an increase in overall mutation frequency following fibre exposure did not achieve statistical significance, the modest increase seen following exposure to erionite and crocidolite is translated into a highly significant change in those components of the spectrum of mutations which result in LOH.