Value of staging squamous cell carcinoma of the anal margin and canal using the sentinel lymph node procedure: an update of the series and a review of the literature.

BACKGROUND: Inguinal metastases in patients affected by anal cancer are an independent prognostic factor for local failure and overall mortality. Since 2001, sentinel lymph node biopsy was applied in these patients. This original study reports an update of personal and previous published series, which were compared with Literature to value the incidence of inguinal metastases T-stage related and the overall incidence of false negative inguinal metastases at sentinel node. METHODS: In all, 63 patients diagnosed with anal cancer submitted to inguinal sentinel node. Furthermore a research in the Pub Med database was performed to find papers regarding this technique. RESULTS: In our series, detection rate was 98.4%. Inguinal metastases were evidentiated in 13 patients (20.6%). Our median follow-up was 35 months. In our series, no false negative nodes were observed. CONCLUSION: Sentinel node technique in the detection of inguinal metastases in patients affected by anal cancer should be considered as a standard of care. It is indicated for all T stages in order to select patients to be submitted to inguinal radiotherapy, avoiding related morbidity in negative ones. An overall 3.7% rate of false negative must be considered acceptable.

Feasibility of the sentinel node biopsy in anal cancer.

AIM: Anal cancer is a rare neoplasm. According to a European Organization for Research and Treatment of Cancer multivariate analysis, synchronous inguinal lymph node metastasis occurs in 10-25% of patients and constitutes an independent prognostic factor for local failure and overall mortality. METHODS: Inguinal lymph node status was assessed using the sentinel node technique in 35 patients with anal cancer. RESULTS: Histology revealed 23 squamous carcinomas, 10 basaloid carcinomas, 1 squamous carcinoma with basaloid areas and 1 spinocellular epithelioma associated with areas of Bowen's disease. Disease stage was T1 in 5 patients, T2 in 18, T3 in 11 and T4 in 1 patient. Lympho-scintigraphy using a GE Millennium gamma camera was performed after peritumoral injection of 37 MBq of 99mTc colloid. Surgical sentinel node biopsy with a portable Scintiprobe MR 100 (Politech, Carsoli, Italy) had a detection rate of 97.1%. Inguinal metastases were detected in 7 (20%) patients, in 2 of which metastasis was bilateral. CONCLUSIONS: Given the correlation between prognosis and node involvement, sentinel node biopsy can be considered a simple method for adequate pretreatment staging of anal carcinoma. Use of the technique could avert the need for prophylactic inguinal radiotherapy in N0-N1 patients, thus reducing the morbidity associated with inguinal radiotherapy. Consistent follow-up is required to evaluate long-term results:

Assessment of AFLP marker behaviour in enriching STS radiation hybrid maps.

Radiation hybrid (RH) mapping provides a powerful tool to build high-resolution maps of genomes. Here, we demonstrate the use of the AFLP technique for high-throughput typing of RH cell lines. Cattle were used as the model species because an RH panel was available to investigate the behaviour of AFLP markers within the microsatellite- and STS-based maps of this species. A total of 747 AFLP markers were typed on the TM112 RH radiation panel and 651 of these were assigned by two-point analysis to the 29 bovine autosomes and sex chromosomes. AFLP markers were added to the 1222 microsatellite and STS markers that were included in earlier RH maps. Multipoint maps were constructed for seven example chromosomes, which retained 248 microsatellite and STS markers, and added 123 AFLP markers at LOD 4. The addition of the AFLP markers increased the number of markers by 42.1% and the map length by 10.4%. The AFLP markers showed lower retention frequency (RF) values than the STS markers. The comparison of RF values in AFLP markers and their corresponding AFLP-derived STSs demonstrated that the lower RF values were due to the lower detection sensitivity of the AFLP technique. Despite these differences, AFLP and AFLP-derived STS markers mapped to identical or similar positions. These results demonstrate that it is possible to merge AFLP and microsatellite markers in the same map. The application of AFLP technology could permit the rapid construction of RH maps in species for which extensive genome information and large numbers of SNP and microsatellite markers are not available.

Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories.

Two pig cell lines derived from kidney and trachea tissues and referred to as newborn swine kidney (NSK) and newborn pig trachea (NPTr) were established following serial culture of primary cells. They were characterized by an epithelial-like morphology, high capacity to replicate and stability of the cell monolayer for several days after seeding. Their modal chromosome number was modified in comparison to that of primary swine cells and they both displayed a transforming potential in vitro and displayed oncogenicity in nude mice. Infection with pig endogenous retroviruses was detected. Almost all the swine viruses tested, i.e., pseudorabies virus, pig parvovirus, hog cholera virus, transmissible gastroenteritis virus of swine, encephalomyocarditis virus, swine vesicular disease virus and the enteroviruses, except pig reproductive respiratory syndrome virus, were capable of replicating in the new cell lines with titres similar to the ones detected in the reference culture systems. Furthermore, all the selected influenza virus sub-types isolated from human, swine and avian species replicated with cytopathic effect in NSK and NPTr cells, whereas, of all the equine influenza viruses tested only the Miami and Suffolk sub-types replicated.

A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease.

Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization.

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

Comparative analysis of calf and cow oocytes during in vitro maturation.

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.