Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy.

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location has an impact on the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the messenger RNA (mRNA) expression of proteins involved in major signaling pathways, including tumor growth (epidermal growth factor receptor (EGFR)), angiogenesis (vascular endothelial growth factor receptor 2 (VEGFR2)), DNA repair (excision repair cross complement group 1 (ERCC1)) and fluoropyrimidine metabolism (thymidylate synthase (TS)). Formalin-fixed paraffin-embedded tumor specimens from 431 advanced CRC patients were analyzed. The presence of seven different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by reverse transcriptase-PCR. BRAF mutations were significantly more common in the proximal colon (P<0.001), whereas KRAS mutations occurred at similar frequencies throughout the colorectum. Rectal cancers had significantly higher ERCC1 and VEGFR2 mRNA levels compared with distal and proximal colon tumors (P=0.001), and increased TS levels compared with distal colon cancers (P=0.02). Mutant KRAS status was associated with lower ERCC1, TS, EGFR and VEGFR2 gene expression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers, as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

KRAS mutations in non-small-cell lung cancer and colorectal cancer: implications for EGFR-targeted therapies.

BACKGROUND: KRAS mutations are associated with diverse biologic functions as well as prognostic and predictive impact in non-small cell-lung cancer (NSCLC) and colorectal cancer (CRC). In CRC, benefit from monoclonal antibody therapies targeting EGFR is generally limited to patients whose tumors have wild-type (WT) KRAS, whereas data suggest that this association is not present for NSCLC. We hypothesized that the unique tobacco-related carcinogenesis of NSCLC results in a divergence of KRAS MT genotype compared with CRC, contributing to differences in outcomes from EGFR-targeted therapies. MATERIAL AND METHODS: Tumor from 2603 patients (838 CRC and 1765 NSCLC) was analyzed for KRAS mutations. DNA was extracted from microdissected formalin-fixed-paraffin-embedded specimens (FFPE) and 7 different base substitutions in codons 12 and 13 of KRAS were determined. RESULTS: KRAS mutation genotype differed significantly between NSCLC and CRC in frequency (25% vs. 39%; p<0.001), smoking-associated G>T transversions (73% versus 27%; p<0.001), and ratio of transversions to transitions (3.5 vs. 0.79; p<0.001). In NSCLC GLY12Cys mutations, resulting from a codon 12 GGT>TGT substitution, were observed in 44% compared to 10% for CRC. In contrast, codon 12 or 13 GLY>ASP substitutions (resulting in a G>A transition) were more frequent in CRC (42%) compared with NSCLC (21%). CONCLUSION: In this large dataset, KRAS mutation patterns are quantitatively and qualitatively distinct between NSCLC and CRC, reflecting in part differences in tobacco-related carcinogenesis. In light of differences in predictive value for EGFR-directed monoclonal antibody therapy and prognosis for specific KRAS mutations between NSCLC and CRC, these data provide an underlying biologic rationale.

Factors specifying cell lineages in the leech.

As in arthropods, several major organ systems in leeches, including body musculature, nervous system and nephridia, are organized into a fixed number of longitudinally iterated units called segments. Many cells, especially neurons, can be uniquely identified from segment to segment. Leech embryos comprise identified cells, which facilitates developmental analysis. So far as it is known, cell lineages in leech are largely determinate. Prior to first cleavage, cytoplasmic reorganization generates domains of yolk-deficient cytoplasm called teloplasm. In situ hybridization experiments suggest that teloplasm is enriched for polyadenylated RNAs. During the first three, unequal cell divisions, teloplasm is segregated to macromere D'; normally, this cell alone cleaves further to generate five bilateral pairs of embryonic stem cells, M, N, O/P and Q teloblasts. Centrifugation experiments have shown a causal link between inheritance of teloplasm and the cleavage pattern that generates teloblasts. Teloblasts undergo highly unequal divisions, generating a longitudinal array of segmental founder cells called m, n, o, p and q blast cells, from which the definitive segmental tissues arise via further stereotyped cell divisions. Microinjecting new-born teloblasts or their precursors with polyadenylic acid induces the formation of supernumerary teloblasts. This discovery permits further analyses of factors specifying the five cell lines generating segmental tissues of the leech.

Developmental and neural regulation of a subsarcolemmal component of the rat neuromuscular junction.

We have generated a monoclonal antibody, designated mAb 3G2, which reacts with a subsarcolemmal component of the neuromuscular junction in adult rats. mAb 3G2 immunoreactivity lies beneath and between the ACh receptor-rich synaptic gutters, around the sole plate nuclei, and at/near sarcomeric Z-disks in the vicinity of the synapse. Localization of mAb 3G2 immunoreactivity to neuromuscular junctions begins postnatally and gradually increases to adult levels. The establishment of this synaptic localization is neurally regulated, as neonatal denervation prevents its occurrence. In adults, denervation results in a loss of synaptic immunoreactivity that returns upon reinnervation. The antigen is also found at the myotendinous junction; its localization here is innervation independent. mAb 3G2 recognizes a 41 kDa protein on immunoblots of extracts of newborn muscle. Based on its distribution within muscle fibers, its developmental and neural regulation, and its molecular weight, the protein recognized by mAb 3G2 can be distinguished from other known postsynaptic proteins. Its neural dependence and developmental regulation suggest that it may participate in synaptic stabilization, perhaps as the intracellular component in a chain of proteins that serve to tether the nerve terminal to the perijunctional region of the muscle fiber.

Differential neural regulation of a neuromuscular junction-associated antigen in muscle fibers and Schwann cells.

Monoclonal antibodies 3G2 and 4E2 recognize a postsynaptic component of rat neuromuscular junctions. In contrast to many other postsynaptic junctional antigens, expression of this antigen is nerve-dependent: immunoreactivity disappears from junctions following denervation and returns upon reinnervation (Astrow et al., 1992 J. Neurosci. 12:1602-1615). Here we show that the epitope is also expressed by Schwann cells and that this expression is also neurally regulated. Weak mAb 3G2/4E2 immunoreactivity was found in myelinating Schwann cells but was not detected in either nonmyelinating Schwann cells or in terminal Schwann cells at the neuromuscular junction. Following axotomy, immunoreactivity increased in myelinating Schwann cells, and nonmyelinating and terminal Schwann cells became immunopositive. Moreover, the immunoreactivity in terminal Schwann cells revealed their extensive sprouting in response to denervation (Reynolds and Woolf, 1992, J. Neurocytol. 21: 50-66). After nerve regeneration, mAb 3G2/4E2 immunoreactivity in all Schwann cells returned towards normal: it disappeared from terminal Schwann cells, returned to low levels in myelinating Schwann cells, and decreased in nonmyelinating Schwann cells. Immunoblots of axotomized nerve and cultured muscle fibers revealed the same set of immunoreactive bands. Therefore, Schwann cells and muscle fibers share the expression of an epitope that is under neural control, but is regulated differently at each site. In Schwann cells, the presence of the nerve suppresses expression of the epitope, whereas in muscle fibers, the nerve terminal promotes this expression. The differential regulation of mAb 3G2/4E2 immunoreactivity in terminal Schwann cells and muscle fibers suggests that the epitope may be involved in interactions between nerve terminals and these cells.

Effects of testosterone on a sexually dimorphic frog muscle: repeated in vivo observations and androgen receptor distribution.

In the present study the sexually dimorphic, androgen-sensitive flexor carpi radialis muscle (FCR) in male Xenopus laevis was viewed repeatedly in vivo to assess the influence of testosterone on muscle fiber size over a period of up to 12 weeks. Regions of the muscle innervated by different spinal nerves responded differently to testosterone treatment. Muscle fibers innervated by spinal nerve 2 (SN2) hypertrophied within 7 days in frogs that had been castrated and given testosterone-filled implants. This initial hypertrophy was followed by a return to normal fiber size a week later, after which fiber size slowly increased again. In castrated males with empty implants, muscle fibers innervated by SN2 gradually atrophied. Fibers innervated by spinal nerve 3 (SN3) were not affected by androgen replacement or withdrawal. The sartorius, a control muscle that is neither sexually dimorphic nor particularly androgen sensitive, was also unaffected. The in vivo observations were confirmed by measurements of muscle fiber cross-sectional areas in frozen sections of whole forelimbs. At 8 and 12 weeks after castration, cross-sectional areas of fibers innervated by SN2 were significantly larger in frogs provided with testosterone than in castrates without testosterone. No difference was found in the SN3 region or in the anconeus caput scapulare (triceps), another control muscle. Immunocytochemistry employing an antibody against the androgen receptor (AR) indicated that the receptor is present in myonuclei of all muscles of the forelimb. While no difference in labeling intensity was detected, the number of AR-containing nuclei per muscle fiber cross-section was higher in fibers innervated by SN2 than in those innervated by SN3, and was yet lower in the triceps. This suggests that regulation of androgen sensitivity may occur via muscle fiber ARs, although an influence of the nerve may also contribute.

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody.

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or "fingers" that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 microm at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.

Precision of reinnervation and synaptic remodeling observed in neuromuscular junctions of living frogs.

Repeated in vivo observations were used to study regenerated nerve terminals in neuromuscular junctions of the adult frog Rana pipiens. Sartorius junctions in living animals were stained with the fluorescent vital dye RH414 and viewed with video fluorescence microscopy. Each junction was observed in the intact muscle and then again 7, 10, and 13 weeks after nerve crush. At 13 weeks, junctions were determined to be mono- or polyneuronally innervated using intracellular recording. Between 7 and 13 weeks, most identified junctions were reinnervated less precisely and completely than described previously. Although some of the original synaptic gutters were reoccupied by regenerated terminal branches, other gutters were only partially occupied, and many appeared abandoned. Junctions showing precise recapitulation of original terminal arborizations comprised a small number of the total examined, as did those where reinnervation was very imprecise. Striking differences in the precision of reinnervation were found within the muscle such that distal terminals regenerated more precisely and completely than did proximal terminals. Terminals in reinnervated muscles were more dynamic than terminals in unoperated muscles over equivalent times. In singly innervated junctions, terminal growth was favored over regression. In doubly innervated junctions, regressive events were more common. Imprecise reinnervation is explained in terms of multisite innervation of muscle fibers and the activity dependence of synaptic stability. We hypothesize that when axons reinnervate the second or third junctions on a fiber, they do so less precisely, because the activity restored by reinnervation of the first junction renders later sites less attractive or less stable.

The origin and selective innervation of early muscle fiber types in the rat.

The diversity of muscle fiber types present in adult animals is present also in the fetus. Fibers generated early and late in fetal development undergo a stereotyped sequence of myosin expressions in giving rise to these fiber types. The differentiation of these fetal fiber types does not require innervation. However, evidence obtained from experiments identifying the types of fibers innervated by single motors suggests that the nervous system comes to recognize this diversity, at least during early postnatal life. Reinnervation experiments suggest that this recognition can occur in the absence of the timing cues normally present in the genesis of fiber types. Thus, a selective innervation of muscle fiber types occurs during development. The role of rearrangement of initial synaptic connections in generating this selectivity is discussed.

Animal and vegetal teloplasms mix in the early embryo of the leech, Helobdella triserialis.

In embryos of the glossiphoniid leech, Helobdella triserialis, as in many annelids, cytoplasmic reorganization prior to first cleavage generates distinct animal and vegetal domains of yolk-deficient cytoplasm, called teloplasm. Both domains are sequestered to the D' macromere, progenitor of the definitive segmental tissues, during the first three rounds of cell division. And it has been believed that during the fourth round of cell division, the obliquely equatorial cleavage of macromere D' cleanly segregates animal teloplasm into an ectodermal precursor, cell DNOPQ, and vegetal teloplasm into a mesodermal precursor, cell DM. But here we report a hitherto unobserved cytoplasmic rearrangement between the second and the fourth divisions that seems to mix the animal and vegetal domains of teloplasm. The newly observed rearrangement consists of the movement of vegetal teloplasm toward the animal pole of cell D' between the second and the fourth cell divisions. Animal and vegetal teloplasms form a single pool of teloplasm in cell D' which is then divided between DM and DNOPQ at the fourth division. The movement of teloplasm was inferred by examination of embryos fixed and sectioned between the second and the fourth rounds of cleavage and was confirmed in living embryos microinjected with rhodamine 123, a fluorescent mitochondrial stain.

Teloplasm formation in a leech, Helobdella triserialis, is a microtubule-dependent process.

Fertilized eggs of the leech Helobdella triserialis undergo a cytoplasmic reorganization which generates domains of nonyolky cytoplasm, called teloplasm, at the animal and vegetal poles. The segregation of teloplasm to one cell of the eight-cell embryo is responsible for a unique developmental fate of that cell, i.e., to give rise to segmental ectoderm and mesoderm. We have studied the cytoplasmic movements that generate teloplasm using time-lapse video microscopy; the formation and migration of rings of nonyolky cytoplasm were visualized using transmitted light, while the movements of mitochondria into these rings were monitored with epifluorescence after labeling embryos with rhodamine 123, a fluorescent mitochondrial dye. To examine the likelihood that cytoskeletal elements play a role in the mechanism of teloplasm formation in Helobdella, we examined the distribution of microtubules and microfilaments during the first cell cycle by indirect immunofluorescence and rhodamine-phalloidin labeling, respectively. The cortex of the early embryo contained a network of microtubules many of which were oriented parallel to the cell surface. As teloplasm formation ensued, microtubule networks became concentrated in the animal and the vegetal cortex relative to the equatorial cortex. More extensive microtubule arrays were found within the rings of teloplasm. Actin filaments appeared in the form of narrow rings in the cortex, but these varied apparently randomly from embryo to embryo in terms of number, size, and position. The role of microtubules and microfilaments in teloplasm formation was tested using depolymerizing agents. Teloplasm formation was blocked by microtubule inhibitors, but not by microfilament inhibitors. These results differ significantly from those obtained in embryos of the oligochaete Tubifex hattai, suggesting that the presumably homologous cytoplasmic reorganizations seen in these two annelids have different cytoskeletal dependencies.

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves.

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cell at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.

Immunohistochemical detection of ornithine-decarboxylase in primary and metastatic human breast cancer specimens.

Increased ornithine decarboxylase (ODC) activity in human breast cancer specimens has recently been shown to be an independent adverse prognostic factor for recurrence and death. Biochemical measurement of ODC, however, is not practical for routine clinical use. Furthermore, it does not take into account the heterogeneous composition of human breast cancers which contain variable proportions of epithelial and stromal elements. Therefore, we developed an immunohistochemical method for ODC determination which can be applied to formalin-fixed, paraffin-embedded tissue sections. We report here our results in a series of 30 human breast cancer samples. ODC expression was detected most consistently in the malignant epithelial component of the tumors. Twenty-seven of 30 samples stained positive with intensities ranging from 1+ to 3+. The fraction of malignant epithelial cells expressing ODC varied among specimens between 10% and > 90%. When quantitated by H-SCORE, ODC expression was significantly higher in the malignant epithelial component than in normal appearing epithelial cells and stroma admixed within the tumor. Normal mammary tissue adjacent to the cancer was available for analysis in six cases. ODC expression was absent in two (while both cancers were positive) but present in four to a degree which was overall comparable to that observed in the corresponding tumors. We believe that this technique will be useful for future studies aimed at expanding our knowledge of the role of ODC and polyamines (PA) in breast cancer biology.