Tumour markers as prognostic factors of survival of gastric cancer patients

The incidence of gastric cancer in the last decades has declined rapidly in the industrialised countries. Worldwide, however, gastric cancer is still the second most common cause of cancer death. Although surgery is currently the most effective treatment, the rapid progress in adjuvant chemotherapy and radiation therapy requires a re-evaluation of prognosis assessment. The TNM staging system of the UICC is ubiquitously used; it groups patients by decreasing survival times from stage I to stage IV based on the spread of disease, i.e. depth of tumour penetration (T), extent of spread to lymph nodes (N), and the presence or absence of distant (M) metastases. This is by far the most consistent prognostic classification system today. However, even within the stage groups there are patients that follow a varying course of disease. Our knowledge of the molecular differences between tumours of the same stage and morphology has been accumulating over the years and methods for a more accurate assessment of the phenotype of neoplasias are of value when evaluating the prognosis of individual patients with gastric cancer. In this study, the immunohistochemical expression of tumour markers involved in different phases in tumourigenesis was examined. The aim was to find new markers which could provide prognostic information in addition to what is provided by the TNM variables. A total of 337 specimens from the primary tumour of patients who underwent surgery for gastric cancer were collected and the immunohistochemical expression of seven different biomarkers was analysed. DNA ploidy and S-phase fraction (SPF) was assessed by flow cytometry. Finally, all biomarkers and clinicopathological prognostic factors were combined and evaluated by a multivariate Cox regression model to elucidate which specific factors provide independent prognostic information. By univariate survival analysis the following variables were significant prognostic factors: epithelial and stromal syndecan-1 expression, stromal tenascin-C expression, expression of tumour-associated trypsin inhibitor (TATI) in cancer cells, nuclear p53 expression, nuclear p21 expression, DNA ploidy, and SPF. By multivariate survival analysis adjusted for all available clinicopathological and biomolecular variables, p53 expression, p21 expression, and DNA ploidy emerged as independent prognostic biomarkers, together with penetration depth of the tumour, presence of nodal metastases, surgical cure of the cancer, and age of the patient at the time of diagnosis.

Detection of renal dysfunction during and after anaesthesia and surgery - evaluation of the influence of inorganic fluoride, ketorolac and clonidine

Drugs and surgical techniques may have harmful renal effects during the perioperative period. Traditional biomarkers are often insensitive to minor renal changes, but novel biomarkers may more accurately detect disturbances in glomerular and tubular function and integrity. The purpose of this study was first, to evaluate the renal effects of ketorolac and clonidine during inhalation anesthesia with sevoflurane and isoflurane, and secondly, to evaluate the effect of tobacco smoking on the production of inorganic fluoride (F-) following enflurane and sevoflurane anesthesia as well as to determine the effect of F- on renal function and cellular integrity in surgical patients. A total of 143 patients undergoing either conventional (n = 75) or endoscopic (n = 68) inpatient surgery were enrolled in four studies. The ketorolac and clonidine studies were prospective, randomized, placebo controlled and double-blinded, while the cigarette smoking studies were prospective cohort studies with two parallel groups. As a sign of proximal tubular deterioration, a similar transient increase in urine N-acetyl-beta-D-glucosaminidase/creatinine (U-NAG/crea) was noted in both the ketorolac group and in the controls (baseline vs. at two hours of anesthesia, p = 0.015) with a 3.3 minimum alveolar concentration hour sevoflurane anesthesia. Uncorrelated U-NAG increased above the maximum concentration measured from healthy volunteers (6.1 units/l) in 5/15 patients with ketorolac and in none of the controls (p = 0.042). As a sign of proximal tubular deterioration, U-glutathione transferase-alpha/crea (U-GST-alpha/crea) increased in both groups at two hours after anesthesia but a more significant increase was noted in the patients with ketorolac. U-GST-alpha/crea increased above the maximum ratio measured from healthy volunteers in 7/15 patients with ketorolac and in 3/15 controls. Clonidine diminished the activation of the renin-angiotensin aldosterone system during pneumoperitoneum; urine output was better preserved in the patients treated with clonidine (1/15 patients developed oliguria) than in the controls (8/15 developed oliguria (p=0.005)). Most patients with pneumoperitoneum and isoflurane anesthesia developed a transient proximal tubular deterioration, as U-NAG increased above 6.1 units/L in 11/15 patients with clonidine and in 7/15 controls. In the patients receiving clonidine treatment, the median of U-NAG/crea was higher than in the controls at 60 minutes of pneumoperitoneum (p = 0.01), suggesting that clonidine seems to worsen proximal tubular deterioration. Smoking induced the metabolism of enflurane, but the renal function remained intact in both the smokers and the non-smokers with enflurane anesthesia. On the contrary, smoking did not induce sevoflurane metabolism, but glomerular function decreased in 4/25 non-smokers and in 7/25 smokers with sevoflurane anesthesia. All five patients with S-F- ≥ 40 micromol/L, but only 6/45 with S-F- less than 40 micromol/L (p = 0.001), developed a S-tumor associated trypsin inhibitor concentration above 3 nmol/L as a sign of glomerular dysfunction. As a sign of proximal tubulus deterioration, U-beta 2-microglobulin increased in 2/5 patients with S-F- over 40 micromol/L compared to 2/45 patients with the highest S-F- less than 40 micromol/L (p = 0.005). To conclude, sevoflurane anesthesia may cause a transient proximal tubular deterioration which may be worsened by a co-administration of ketorolac. Clonidine premedication prevents the activation of the renin-angiotensin aldosterone system and preserves normal urine output, but may be harmful for proximal tubules during pneumoperitoneum. Smoking induces the metabolism of enflurane but not that of sevoflurane. Serum F- of 40 micromol/L or higher may induce glomerular dysfunction and proximal tubulus deterioration in patients with sevoflurane anesthesia. The novel renal biomarkers warrant further studies in order to establish reference values for surgical patients having inhalation anesthesia.

Inflammation-induced atherogenesis, liver alterations, and cardiovascular outcome

Cardiovascular diseases, which presently are considered inflammatory diseases, affect millions of people worldwide. Chronic infections may contribute to the systemic inflammation suggested to increase the risk for cardiovascular diseases. Such chronic infections are periodontitis and Chlamydia pneumoniae infection. They are highly prevalent as approximately 10% of adult population and 30% of people over 50 years old are affected by severe periodontitis and 70-80% of elderly people are seropositive for C. pneumoniae. Our general aim was to investigate the role of infection and inflammation in atherosclerosis both in animal and human studies. We aimed to determine how the two pathogens alter the atherosclerosis-associated parameters, and how they affect the liver inflammation and lipid composition. Furthermore, we evaluated the association between matrix metalloproteinase-8 (MMP-8), a proteinase playing a major role in inflammation, and the future cardiovascular diseases (CVD) events in a population-based cohort. For the animal experiments, we used atherosclerosis-susceptible apolipoprotein E deficient (apoE-/-) mice. They were kept in germ free conditions and fed with a normal chow diet. The bacteria were administered either intravenously (A. actinomycetemcomitans) or intranasally (C. pneumoniae). Several factors were determined from serum as well as from aortic and hepatic tissues. We also determined how cholesterol efflux, a major event in the removal of excess cholesterol from the tissues, and endothelial function were affected by these pathogens. In the human study, serum MMP-8 and its tissue inhibitor (TIMP-1) concentrations were measured and their associations during the follow-up time of 10 years with CVD events were determined. An infection with A. actinomycetemcomitans increased concentrations of inflammatory mediators, MMP production, and cholesterol deposit in macrophages, decreased lipoprotein particle size, and induced liver inflammation. C. pneumoniae infection also elicited an inflammatory response and endothelial dysfunction, as well as induced liver inflammation, microvesicular appearance and altered fatty acid profile. In the population-based cohort, men with increased serum MMP-8 concentration together with subclinical atherosclerosis (carotid artery intima media thickness > 1mm) had a three-fold increased risk for CVD death during the follow-up. The results show that infections with A. actinomycetemcomitans and C. pneumoniae induce proatherogenic changes, as well as affect the liver. These data therefore support the concept that common infections have systemic effects and could be considered as cardiovascular risk factors. Furthermore, our data indicate that, as an independent predictor of fatal CVD event, serum MMP-8 could have a clinical significance in diagnosing cardiovascular diseases.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Human trypsinogens in the pancreas and in cancer

Human pancreatic juice contains two major trypsinogen isoenzymes called trypsinogen-1 and -2, or cationic and anionic trypsinogen, respectively. Trypsinogen isoenzymes are also expressed in various normal and malignant tissues. We aimed at developing monoclonal antibodies (MAbs) and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these MAbs and methods we purified, characterized and quantitated trypsinogen isoenzymes in serum samples, ovarian cyst fluids and conditioned cell culture media. In sera from healthy subjects and patients with extrapancreatic disease the concentration of trypsinogen-1 is higher than that of trypsinogen-2. However, in acute pancreatitis we found that the concentration of serum trypsinogen-2 is 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration is only 15-fold. This suggested that trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis. In human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme. Most notably, in mucinous cyst fluids the levels of TAT-2 were higher in borderline and malignant than in benign cases. The increased levels in association with malignancy suggested that TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2. Trypsinogen-1 was detected in only one of nine samples. These results suggested that the expression of trypsinogen is not restricted to the pancreas. Determination of the isoenzyme pattern by ion exchange chromatography revealed isoelectric variants of trypsinogen isoenzymes in serum samples. Intact trypsinogen isoenzymes and tryptic and chymotryptic trypsinogen peptides were purified and characterized by mass spectrometry, Western blot analysis and N-terminal sequencing. The results showed that pancreatic trypsinogen-1 and -2 are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin, thus Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.

Complex trait of acute pancreatitis : Studies of candidate genes and adipokines

Acute pancreatitis (AP) is a common disease. Mild disease resolves spontaneously in a few days. Severe forms of the disease can lead to local complications, necrosis, and abscesses in and around the pancreas. Systemic inflammation in severe AP is associated with distant organ failures. The aim of this study is to identify genetically determined prognostic factors involved in the clinical features of AP. The study employs a candidate-gene approach, and the genes are involved in trysinogen activation in the initiation phase of the disease, as well as in the systemic inflammation as the disease proceeds. The last study examines adipokines, fat-derived hormones characterized with the capacity to modify inflammation. SPINK 1 is a gene coding trypsin activation inhibitor. Mutations N34S and P55N were determined by minisequencing methods in 371 AP patients and in 459 controls. The mutation N34S was more common in AP patients (7.8%) than in controls (2.6%). This suggests that SPINK 1 gene mutation N34S is a risk factor for AP. In the fourth study, in 12 matched pairs of patients with severe and mild AP, levels of adipokines, adiponectin, and leptin were evaluated. Plasma adipokine levels did not differ between patients with mild and severe AP. The results suggest that in AP, adipokine plasma levels are not factors predisposing to organ failures. This study identified the SPINK 1 mutation N34S to be a risk factor for AP in the general population. As AP is a multifactorial disease, and extensive genetic heterogeneity is likely, further identification of genetic factors in the disease requires larger future studies with more advanced genetic study models. Further identification of the patient characteristics associated with organ failures offers another direction of the study to achieve more detailed understanding of the severe form of AP.

Thrombophilia and direct thrombin inhibitor lepirudin clinical and monitoring aspects

Thrombophilia (TF) predisposes both to venous and arterial thrombosis at a young age. TF may also impact the thrombosis or stenosis of hemodialysis (HD) vascular access in patients with end-stage renal disease (ESRD). When involved in severe thrombosis TF may associate with inappropriate response to anticoagulation. Lepirudin, a potent direct thrombin inhibitor (DTI), indicated for heparin-induced thrombocytopenia-related thrombosis, could offer a treatment alternative in TF. Monitoring of narrow-ranged lepirudin demands new insights also in laboratory. The above issues constitute the targets in this thesis. We evaluated the prevalence of TF in patients with ESRD and its impact upon thrombosis- or stenosis-free survival of the vascular access. Altogether 237 ESRD patients were prospectively screened for TF and thrombogenic risk factors prior to HD access surgery in 2002-2004 (mean follow-up of 3.6 years). TF was evident in 43 (18%) of the ESRD patients, more often in males (23 vs. 9%, p=0.009). Known gene mutations of FV Leiden and FII G20210A occurred in 4%. Vascular access sufficiently matured in 226 (95%). The 1-year thrombosis- and stenosis-free access survival was 72%. Female gender (hazards ratio, HR, 2.5; 95% CI 1.6-3.9) and TF (HR 1.9, 95% CI 1.1-3.3) were independent risk factors for the shortened thrombosis- and stenosis-free survival. Additionally, TF or thrombogenic background was found in relatively young patients having severe thrombosis either in hepatic veins (Budd-Chiari syndrome, BCS, one patient) or inoperable critical limb ischemia (CLI, six patients). Lepirudin was evaluated in an off-label setting in the severe thrombosis after inefficacious traditional anticoagulation without other treatment options except severe invasive procedures, such as lower extremity amputation. Lepirudin treatments were repeatedly monitored clinically and with laboratory assessments (e.g. activated partial thromboplastin time, APTT). Our preliminary studies with lepirudin in thrombotic calamities appeared safe, and no bleeds occurred. An effective DTI lepirudin calmed thrombosis as all patients gradually recovered. Only one limb amputation was performed 3 years later during the follow-up (mean 4 years). Furthermore, we aimed to overcome the limitations of APTT and confounding effects of warfarin (INR of 1.5-3.9) and lupus anticoagulant (LA). Lepirudin responses were assessed in vitro by five specific laboratory methods. Ecarin chromogenic assay (ECA) or anti-Factor IIa (anti-FIIa) correlated precisely (r=0.99) with each other and with spiked lepirudin in all plasma pools: normal, warfarin, and LA-containing plasma. In contrast, in the presence of warfarin and LA both APTT and prothrombinase-induced clotting time (PiCT®) were limited by non-linear and imprecise dose responses. As a global coagulation test APTT is useful in parallel to the precise chromogenic methods ECA or Anti-FIIa in challenging clinical situations. Lepirudin treatment requires multidisciplinary approach to ensure appropriate patient selection, interpretation of laboratory monitoring, and treatment safety. TF seemed to be associated with complicated thrombotic events, in venous (BCS), arterial (CLI), and vascular access systems. TF screening should be aimed to patients with repeated access complications or prior unprovoked thromboembolic events. Lepirudin inhibits free and clot-bound thrombin which heparin fails to inhibit. Lepirudin seems to offer a potent and safe option for treatment of severe thrombosis. Multi-centered randomized trials are necessary to assess the possible management of complicated thrombotic events with DTIs like lepirudin and seek prevention options against access complications.

Role of γ1 Laminin and Its KDI Peptide in the Central Nervous System

Since the 1980 s, laminin-1 has been linked to regeneration of the central nervous system (CNS) and promotion of neuronal migration and axon guidance during CNS development. In this thesis, we clarify the role of γ1 laminin and its KDI tripeptide in development of human embryonic spinal cord, in regeneration of adult rat spinal cord injury (SCI), in kainic acid-induced neuronal death, and in the spinal cord tissue of amyotrophic lateral sclerosis (ALS). We demonstrated that γ1 laminin together with α1, β1, and β3 laminins localize at the floor plate region in human embryonic spinal cord. This localization of γ1 laminin is in spatial and temporal correlation with development of the spinal cord and indicates that γ1 laminin may participate in commissural axon guidance during the embryonic development of the human CNS. With in vitro studies using the Matrigel culture system, we demonstrated that the KDI tripeptide of γ1 laminin provides a chemotrophic guidance cue for neurites of the human embryonic dorsal spinal cord, verifying the functional ability of γ1 laminin to guide commissural axons. Results from our experimental SCI model demonstrate that the KDI tripeptide enhanced functional recovery and promoted neurite outgrowth across the mechanically injured area in the adult rat spinal cord. Furthermore, our findings indicate that the KDI tripeptide as a non-competitive inhibitor of the ionotropic glutamate receptors can provide when administered in adequate concentrations an effective method to protect neurons against glutamate-induced excitotoxic cell death. Human postmortem samples were used to study motor neuron disease, ALS (IV), and the study revealed that in human ALS spinal cord, γ1 laminin was selectively over-expressed by reactive astrocytes, and that this over-expression may correlate with disease severity. The multiple ways by which γ1 laminin and its KDI tripeptide provide neurotrophic protection and enhance neuronal viability suggest that the over-expression of γ1 laminin may be a glial attempt to provide protection for neurons against ALS pathology. The KDI tripeptide is effective therapeutically thus far in animal models only. However, because KDI containing γ1 laminin exists naturally in the human CNS, KDI therapies are unlikely to be toxic or allergenic. Results from our animal models are encouraging, with no toxic side-effects detected even at high concentrations, but the ultimate confirmation can be achieved only after clinical trials. More research is still needed until the KDI tripeptide is refined into a clinically applicable method to treat various neurological disorders.

Modulation of growth by aromatase inhibitor treatment in boys: Efficacy and safety

Without estrogen action, the fusion of the growth plates is postponed and statural growth continues for an exceptionally long time. Aromatase inhibitors, blockers of estrogen biosynthesis, have therefore emerged as a new potential option for the treatment of children with short stature. We investigated the efficacy of the aromatase inhibitor letrozole in the treatment of boys with idiopathic short stature (ISS) using a randomised, placebo-controlled, double-blind research setting. A total of 30 boys completed the two-year treatment. By decreasing estrogen-mediated central negative feedback, letrozole increased gonadotrophin and testosterone secretion in pubertal boys, whereas the pubertal increase in IGF-I was inhibited. Treatment with letrozole effectively delayed bone maturation and increased predicted adult height by 5.9 cm (P0.001), while placebo had no effect on either parameter. The effect of letrozole treatment on near-final height was studied in another population, in boys with constitutional delay of puberty, who received letrozole (n=9) or placebo (n=8) for one year, in combination with low-dose testosterone for six months during adolescence. The mean near-final height of boys randomised to receive testosterone and letrozole was significantly greater than that of boys who received testosterone and placebo (175.8 vs. 169.1 cm, P=0.04). As regards safety, treatment effects on bone health, lipid metabolism, insulin sensitivity, and body composition were monitored in boys with ISS. During treatment, no differences in bone mass accrual were evident between the treatment groups, as evaluated by dual-energy x-ray absorptiometry measurements of the lumbar spine and femoral neck. Bone turnover and cortical bone growth, however, were affected by letrozole treatment. As indicated by differences in markers of bone resorption (U-INTP) and formation (S-PINP and S-ALP), the long-term rate of bone turnover was lower in letrozole-treated boys, despite their more rapid advancement in puberty. Letrozole stimulated cortical bone growth in those who progressed in puberty: the metacarpal index (MCI), a measure of cortical bone thickness, increased more in letrozole-treated pubertal boys than in placebo-treated pubertal boys (25% vs. 9%, P=0.007). The change in MCI correlated positively with the mean testosterone-to-estradiol ratio. In post-treatment radiographic evaluation of the spine, a high rate of vertebral deformities - mild anterior wedging and mild compression deformities - were found in both placebo and letrozole groups. In pubertal boys with ISS treated with letrozole, stimulated testosterone secretion was associated with a decrease in the percentage of fat mass and in HDL-cholesterol, while LDL-cholesterol and triglycerides remained unchanged. Insulin sensitivity, as evaluated by HOMA-IR, was not significantly affected by the treatment. In summary, treatment with the aromatase inhibitor letrozole effectively delayed bone maturation and increased predicted adult height in boys with ISS. Long-term follow-up data of boys with constitutional delay of puberty, treated with letrozole for one year during adolescence, suggest that the achieved gain in predicted adult height also results in increased adult height. However, until the safety of aromatase inhibitor treatment in children and adolescents is confirmed, such treatment should be considered experimental.