[Haematologic parameters in opiate addicts]. / Parámetros hematológicos en pacientes opiaceodependientes.

Levels of haemoglobin, haematocrit, number of red cells and leukocytes, haematological index (MCV, MCH, MCHC, DEI, MPV), sedimentation rate, iron, parameter related to accumulation and transport of iron (transferrin, ferritin, iron binding capability, transferring saturation index) and platelets were determined in blood samples of opiate addicts and they were compared with these results obtained in a control group. For both sexes, the levels of haemoglobin, haematocrit, iron and platelets were similar in the control groups and opiate addicts. Opiate addicts females presented a number of red cells and platelets higher and lower respectively than the correspond values of the control group. The haematological index in opiate addicts were higher than the values in the control groups for overall and considering males and females independently. The levels of ferritin in opiate addicts males were higher than these levels found in control groups, occurring the contrary in the females. Opiate addicts males and females included in the methadone maintenance treatment program (MMTP) presented similar values of haemoglobin, haematocrit, number of red cells and leukocytes than the corresponding opiate addicts non-included in MMTP, except opiate addicts males, who showed lower haematocrit values.

Virtually 100% of melanoma cell lines harbor alterations at the DNA level within CDKN2A, CDKN2B, or one of their downstream targets.

The cyclin-dependent kinase inhibitor 2A (CDKN2A), or p16INK4a, gene on 9p21 is important in the genesis of both familial and sporadic melanoma. Homozygous deletions and intragenic mutations of this gene have been identified in both melanoma cell lines and uncultured tumors, although the frequency of these alterations is higher in the cell lines. A proportion of melanoma cell lines and tumors without deletion/mutation of CDKN2A have also been determined to harbor transcriptionally inactive CDKN2A alleles or carry alterations in other components of the pathway through which p16INK4a acts on pRb to mediate cell cycle arrest. We sought to determine the frequency of these alternative events (in relationship to those that specifically inactivate CDKN2A) in a panel of 45 melanoma cell lines. Surprisingly, at the DNA level alone, 96% (43/45) of melanoma cell lines examined were found to be deleted/mutated/methylated for CDKN2A (34/45), homozygously deleted for CDKN2A's neighbor and homolog CDKN2B (6/45), and/or mutated/amplified for CDK4 (5/45). In two of these 43 cases, homozygous deletions of CDKN2A were detected along with a CDK4 mutation or amplification of the cyclin D1 (CCND1) gene. The latter discoveries were made in two of three cell lines which harbored extremely large (3-6 Mb) homozygous deletions on 9p21; all other homozygous deletions in similarly affected cell lines (N = 23) were confined to a region immediately surrounding the CDKN2A/CDKN2B loci. These results suggest that (1) only melanoma cells with alterations in this pathway can be propagated in culture, and (2) the homozygous deletions on 9p21 in the cell lines, which are also mutated/amplified for CDK4 or CCND1, could serve to target tumor suppressor genes other than CDKN2A.

Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors.

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds.

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition.

Loss of the p16INK4a and p15INK4b genes, as well as neighboring 9p21 markers, in sporadic melanoma.

Although homozygous deletions of the cyclin-dependent kinase inhibitor 2 gene p16INK4a on 9p21 have been reported frequently in metastatic melanoma cell lines, and intragenic mutations within the p16INK4a gene have been detected in familial melanoma kindreds, specific targeting of this gene in the development of sporadic melanoma in vivo remains controversial. Southern analyses were performed in this study to initially assess the frequency of hemi- or homozygous losses of p16INK4a, as well as its neighboring family member, p15INK4b, and other candidate regions within 9p21, in sporadic melanoma. Overall, 22 of 40 (55%) uncultured sporadic melanoma DNAs were determined to harbor deletions of 1-11 markers/genes located on 9p21. This included 10 tumors (25%; 10 of 40) with homozygous deletions limited to either the p16INK4a gene only (20%; 2 of 10), both the p16INK4a and p15INK4b genes (10%; 1 of 10), another novel 9p21 gene, FB19 (10%; 1 of 10), or all three of these genes plus surrounding markers (60%; 6 of 10). In subsequent single-strand conformation polymorphism and sequencing analyses, intragenic mutations in the p16INK4a gene were also revealed in two (10%; 2 of 21) melanoma DNAs that retained one copy of this locus. By comparison, the frequency of pl6INK4a and p15INK4b homozygous deletions, as well as p16INK4a mutations, in melanoma cell lines (analyzed in parallel) was 2-3-fold higher at 61 (23 of 38) and 24% (9 of 38), respectively. These findings indicate that (a) p16INK4a is inactivated in vivo in over one-fourth (27.5%; 11 of 40) of sporadic melanomas; (b) mutation/deletion of p16INK4a may confer a selective growth advantage in vitro; and (c) other 9p21 tumor suppressor genes could be targeted during the development of melanoma.

Homozygous loss of the p15INK4B gene (and not the p16INK4 gene) during tumor progression in a sporadic melanoma patient.

Homozygous deletions of 9p21, including the cyclin-dependent kinase inhibitor genes p16INK4 and p15INK4B, have been reported frequently in melanoma (as well as other tumor) cell lines. Germline mutations within the p16INK4 gene have also been described in a proportion of familial melanoma kindreds, suggesting that p16INK4 is the 9p21 "melanoma" gene. We have previously concluded that deletion of this chromosomal region can occur early (before metastasis) and in vivo in sporadic melanoma due to the identification of identical hemizygous losses on 9p21 in six autologous melanoma cell lines established from an individual patient (DX). These related cell lines have now been used to evaluate the timing of deletion/mutation of the p16INK4 and p15INK4B genes during tumor progression in melanoma. Surprisingly, homozygous deletions of a < or = 200-kb region surrounding p15INK4B, but not p16INK4, were detected in all six cell lines. Furthermore, single strand conformation polymorphism and sequencing analysis of the remaining p16INK4 allele in each case revealed only one intragenic mutation (in DX-6), whereas Western analysis provided evidence that p16INK4 protein was expressed in all six instances. These findings, taken together with those generated on other unrelated melanoma tumors and cell lines, suggest that hemizygous loss (or haplo-insufficiency) of the p16INK4 gene may be enough to place a melanocyte on a tumor pathway, and/or that the p16INK4 gene is not the sole 9p21 locus targeted in sporadic melanoma.

Mutations of the CDKN2/p16INK4 gene in Australian melanoma kindreds.

The cyclin dependent kinase inhibitor 2 (CDKN2) gene on chromosome 9p21 is potentially involved in the genesis of many cancers and is currently under intense investigation as a possible melanoma susceptibility locus. We have analyzed 18 Australian melanoma kindreds for mutations within the coding and neighboring splice junction portions of the CDKN2 gene. In seven kindreds (including our six largest), CDKN2 mutations were found to segregate with the putative melanoma chromosome previously assigned by 9p haplotype analysis. These changes included the duplication of a 24 bp repeat, a deleted C residue resulting in the introduction of a premature stop codon, and four single basepair changes causing amino acid substitutions. Mutations segregated to 46 of 51 affected individuals in these seven kindreds, with three apparent sporadic cases in one family and one in each of another two families. Penetrance was variable (55-100%) among the different mutations. These data provide additional strong support that the CDKN2 gene is the chromosome 9p21 familial melanoma locus.

Loss of expression of the p16/cyclin-dependent kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with invasive stage of tumor progression.

Sporadic and familial malignant melanoma susceptibility has been linked to defects in the chromosomal region 9p21. Recently, a putative 9p21 tumor suppressor gene, the cyclin dependent kinase inhibitor 2 (CDKN2) or p16 gene, has been shown to be deleted, mutated, or rearranged in a high percentage of sporadic melanoma cell lines, as well as mutated in the germline of a proportion of familial melanoma patients. CDKN2 encodes a M(r) 16,000 protein (p16) that plays a key role in cell cycle control by binding to the cyclin-dependent kinase 4 enzyme and inhibiting its ability to phosphorylate critical substrates necessary for transition past the G1 phase of the cell cycle. Thus, mutations or deletions of the CDKN2 gene could result in abnormal proliferation via defective cell cycle control. The correlation of 9p21 cytogenetic and molecular alterations with the clinical stages of melanoma progression suggests that dysfunction of a gene within this chromosomal region is critical to the evolution of melanoma. However, it remains unclear whether this gene is the CDKN2 gene. If so, then loss of p16 is potentially an initiating or early event in melanoma progression. To address the issues of what is the potential involvement of the CDKN2 gene in sporadic melanoma and precisely when during the clinically evident stages of melanoma progression defects in CDKN2 occur, we have evaluated by immunohistochemistry the expression of p16 protein in 103 melanocytic lesions representing all stages in the progression of melanoma. Our results suggest that loss of p16 protein expression is (a) not necessary for tumor initiation in malignant melanoma because all melanomas in situ and the majority of primary invasive melanomas retain expression of this protein; and (b) potentially more related to invasiveness or the ability to metastasize, because 52% of primary invasive tumors and 72% of metastatic lesions show partial or complete loss of expression of p16.

Evaluation of a 24-hour infusion of etidronate disodium for the treatment of hypercalcemia of malignancy.

BACKGROUND: Hypercalcemia is a serious and common complication of malignancy. Etidronate, a known inhibitor of osteoclastic bone resorption, is approved in the therapy of hypercalcemia of malignancy (HCM) at a dose of 7.5 mg/kg/day infused during a period of 2-4 hours on 3 consecutive days. A multicenter study was conducted to evaluate the safety and efficacy of a single 24-hour infusion of etidronate disodium in patients with HCM. METHODS: Selected patients with HCM had disease refractory to at least 24-hours of intravenous fluid (more than 3 l/day) with two albumin-adjusted serum calcium concentrations greater than 11.5 mg/dl drawn 24 hours apart before etidronate treatment. Thirty patients were enrolled; 13 received 25 mg/kg for 24 hours, 12 received 30 mg/kg for 24-hours, 3 received incorrect doses (2 overdoses, and 1 underdose) and 2 died of disease-related complications before day 7. Of the 25 evaluable patients, 15 were men and 10 were women. Median age was 53 years (range, 20-75 years). Twelve patients (6 in each treatment group) had confirmed skeletal metastases. RESULTS: During the week after treatment, the 25 mg/kg group had adjusted serum calcium levels fall from a mean preinfusion baseline of 13.3 +/- 0.3 mg/dl (plus or minus the standard error of the mean) to a mean nadir of 10.9 +/- 0.4 mg/dl (the average of each patient's lowest calcium values). The 30 mg/kg group had adjusted serum calcium levels fall from a mean preinfusion baseline of 13.8 +/- 0.4 mg/dl to a mean nadir of 10.5 +/- 0.3 mg/dl. The average day that nadir occurred was day 5.7 for the 25 mg/kg group and day 5.6 for the 30 mg/kg group. The mean maximum reduction (delta) derived from the patients' nadirs in the 25 mg/kg dose group was 2.5 +/- 0.4 mg/dl and 3.3 +/- 0.3 mg/dl for the 30 mg/kg dose. Time to effect (either a partial response defined as a 15% or greater decrease in the adjusted serum calcium from the preinfusion value or a complete eucalcemic response defined as a reduction to the laboratory's eucalcemic range) occurred on average on day 4.6 in the 25 mg/kg group and day 3.7 in the 30 mg/kg group. Nine of the 13 (69%) patients in the 25 mg/kg treatment group had either partial or complete response to the 24-hour infusion. Five of these patients (38% of the 13 patients) of the 25 mg/kg group had serum calcium levels fall to their laboratory's eucalcemic range before day 7 (a complete response), 4 (31%) had partial response only, and 4 had no response. In the 30 mg/kg group, 11 of 12 (92%) patients had at least partial responses. Eight of the 12 (67%) patients had adjusted serum calcium concentrations fall to the eucalcemic range by day 7, 3 (25%) had a partial response, and 1 had no response. Reported adverse experiences generally were attributable to the underlying disease. The reduction in the serum calcium throughout the week for the 30 mg/kg dose group was significantly greater than that for the 25 mg/kg group (analysis of variance, P < 0.0001). CONCLUSIONS: Etidronate, when administered intravenously at 30 mg/kg during a period of 24 hours, apparently was safe and effective in this study for treatment of hypercalcemia in patients with a wide variety of tumor types. This regimen may offer a more convenient method of administration than does standard etidronate therapy for the treatment of HCM.

Effectiveness of a 24-hour infusion of etidronate disodium in the treatment of hypercalcemia of malignant disease. A dose-ranging pilot study.

A dose-ranging, baseline-controlled study was undertaken to assess the safety and effectiveness of a 24-hour infusion of etidronate disodium in treating patients with hypercalcemia of malignant disease. Patients with hypercalcemia refractory to at least 48 h of saline loading (greater than 3 1/day) with two albumin-adjusted serum calcium values between 11.1 and 12.0 mg/dl or one albumin-adjusted serum calcium greater than 12.0 mg/dl within 48 h of therapy were admitted to the study. A total of 26 patients were treated in a dose-escalating fashion with 5, 10, 15, 20 or 25 mg/kg of intravenous etidronate disodium over 24 h. Patients treated with 5, 10 or 15 mg/kg did not have significant reductions in albumin-adjusted serum calcium during the first 7 days. In the 6 patients who made up the 20 mg/kg group, adjusted serum calcium levels fell from an average of 13.8 +/- 0.5 mg/dl on day 1 before infusion to 11.7 +/- 0.3 mg/dl (p less than 0.05) by day 7. In the 8 patients in the 25 mg/kg group, adjusted serum calcium levels decreased from an average of 12.9 +/- 0.5 mg/dl on day 1 before infusion to 10.9 +/- 0.4 mg/dl (p less than 0.05) by day 7. All 8 patients in the 25 mg/kg group achieved a fall in albumin-adjusted serum calcium to less than 11.1 mg/dl within the 1 week with a minimum decrement of 0.6 mg/dl and a maximum of 5.5 mg/dl.(ABSTRACT TRUNCATED AT 250 WORDS)

Leg size and muscle functions associated with leg compliance.

Leg compliance is "causally related with greater susceptibility" to orthostatic stress. Since peak O2 uptake (peak VO2) and muscle strength may be related to leg compliance, we examined the relationships between leg compliance and factors related to muscle size and physical fitness. Ten healthy men, 25-52 yr, underwent tests for determination of vascular compliance of the calf (Whitney mercury strain gauge), peak VO2 (Bruce treadmill), calf muscle strength (Cybex isokinetic dynamometer), body composition (densitometry), and anthropometric measurements of the calf. Cross-sectional areas (CSA) of muscle, fat, and bone in the calf were determined by computed tomography scans. Leg compliance was not significantly correlated with any variables associated with physical fitness per se (peak VO2, calf strength, age, body weight, or composition). Leg compliance correlated with calf CSA (r = -0.72, P less than 0.02) and calculated calf volume (r = -0.67, P less than 0.03). The most dominant contributing factor to the determination of leg compliance was CSA of calf muscle (r = -0.60, P less than 0.06), whereas fat and bone were poor predictors (r = -0.11 and 0.07, respectively). We suggest that leg compliance is less when there is a large muscle mass providing structural support to limit expansion of the veins. This relationship is independent of aerobic and/or strength fitness level of the individual.