Solitary bronchioloalveolar carcinoma: CT criteria

The computed tomographic (CT) scans of 30 patients with solitary bronchioloalveolar carcinoma were reviewed. Common features at CT included the peripheral or subpleural location of a pulmonary mass (25 cases), pseudocavitation (18 cases), heterogeneous attenuation (17 cases), irregular margins forming a star pattern (22 cases), and pleural tags (21 cases). Using these CT criteria, four independent observers attempted to identify cases of bronchioloalveolar carcinoma from a larger sample of lung cancers and benign lesions by categorizing a series of test cases into four probability categories. Although the bronchioloalveolar carcinomas were correctly ranked in the two highest probability categories 75% of the time (in 45 of 60 cases), there was considerable overlap with other lung lesions, particularly with adenocarcinoma and large cell undifferentiated carcinoma. However, even though the typical features of bronchioloalveolar carcinoma are not invariable or highly specific, they are characteristic enough to suggest the diagnosis.     

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.      

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

Comprehensive mutational scanning of the p53 coding region by two- dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long- distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li- Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.      

Associations between both genetic and environmental biomarkers and lung cancer: evidence of a greater risk of lung cancer in women smokers

This molecular epidemiologic case-control study of lung cancer incorporated three complementary biomarkers: the glutathione S- transferase M1 (GSTM1) null genotype, a potential marker of susceptibility, and polycyclic aromatic hydrocarbon-DNA adducts (PAH- DNA) and sister chromatid exchanges (SCE), both indicators of environmentally induced genetic damage. Associations between biomarkers and lung cancer were investigated, as were possible gene-environment interactions between the GSTM1 null genotype and tobacco smoke exposure. Subjects included 136 primary non-small cell lung cancer surgical patients and 115 controls at the Columbia Presbyterian Medical Center. Questionnaire and Tumor Registry data, pre-treatment blood samples and biomarker measurements on blood were obtained. Overall, GSTM1 null genotype was significantly associated with lung cancer [odds ratio (OR) = 2.04, 95% confidence interval (CI) = 1.13-3.68]. ORs for GSTM1 and lung cancer were significant in females (2.50, 1.09-5.72) and smokers (2.25, 1.11-4.54) and not significant in males (1.4, 0.58-3.38) and non-smokers (0.88, 0.18-4.33). However, ORs for males versus females and smokers versus non-smokers did not differ significantly. The OR for GSTM1 and lung cancer in female smokers was 3.03 (1.09- 8.40), compared with 1.42 (0.53-4.06) in male smokers. In contrast to PAH-DNA adducts in leukocytes, SCE did not differ between cases and controls. Neither biomarker differed significantly between the two GSTM1 genotypes. The combined effect of elevated PAH-DNA adducts and GSTM1 genotype on case-control status (16.19, 1.2-115) appeared multiplicative. Results suggest that the effect of the GSTM1 null genotype is greatest in female smokers, which is consistent with other evidence that indicates that women are at higher risk of lung cancer than males, given equal smoking. Persons with both the GSTM1 deletion and elevated PAH-DNA adducts may represent a sensitive subpopulation with respect to carcinogens in tobacco smoke and other environmental media.      

Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than β-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Mu-opioid receptor (MOR) agonists are the most effective treatments for moderate to severe acute and chronic pain, yet their use is limited by serious side effects, including constipation, respiratory depression, and physical and psychological dependence. These side effects are on-target effects (MOR-mediated) and result from the wide distribution of MORs across the central nervous system (CNS) (1, 2). Safer pain therapies are desperately needed. However, because of the efficacy of MOR agonists in blocking pain, this receptor continues to be a primary target for the discovery of novel pain therapies. Unfortunately, most drug discovery programs involve designing compounds that bind to the orthosteric site on MOR—the site that binds endogenous opioid peptides as well as exogenous opioids. Not surprisingly, these newer drugs tend to exhibit qualitatively similar side effect profiles to traditional opioid analgesics.As an alternative, we have discovered small molecule, positive allosteric modulators of MOR [mu-PAMs (3)], including BMS-986122 (SI Appendix, Fig. S1). Such compounds interact with a site on MOR that is spatially distinct from the orthosteric site (3–7). Across a variety of in vitro assays, mu-PAMs increase the affinity and/or potency of orthosteric agonists at MOR, including exogenous MOR agonists as well as the endogenous opioid peptides Leucine- and Methionine-enkephalin, endomorphin-1, and β-endorphin (3, 8).These in vitro studies have led to development of a so-far untested hypothesis that in vivo, mu-PAMs will promote the activity of endogenous opioid peptides released during pain (9–11). If this hypothesis is correct, mu-PAMs could replace traditional opioids by boosting the body’s own natural response to pain to provide clinically meaningful analgesia. In support of this concept, so called “enkephalinase inhibitors” that prolong the lifetime of endogenous opioid peptides are effective in the management of pain in preclinical and clinical studies (12–14), although such compounds are not selective for opioid peptides. Since mu-PAMs do not alter peptide release or metabolism, they should be more selective than enkephalinase inhibitors and also preserve the natural spatial and temporal release of the peptides in vivo following injury and/or during pain. To test this hypothesis, we examined the antinociceptive effects of BMS-986122 in mouse models of acute and inflammatory pain using measures of pain-evoked and pain-depressed behaviors as well as opioid side effects and the potential role of endogenous opioid peptides in these responses.     

A diaphragmatic retroperitoneal cyst

Diaphragmatic lesions are usually congenital bronchogenic cysts. A patient with a known diaphragmatic cyst presented with new onset right upper quadrant pain. Repeat imaging showed enlargement of the cyst, the CA19–9 cancer marker was raised at 312iu/ml (normal: <27iu/ml) and positron emission tomography combined with computed tomography showed focally increased uptake in the cystic wall. In view of symptoms and risk of neoplasia, the lesion was excised. Histology showed a benign epidermoid cyst. Features falsely suggesting neoplasia have been reported previously with benign splenic cysts but not with a benign diaphragmatic epidermoid cyst.