HER2-low expression in patients with advanced or metastatic solid tumors.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-low is a newly defined category with HER2 1+ or 2+ expression by immunohistochemistry (IHC) and lack of HER2 gene amplification measured by in situ hybridization (ISH). Much remains unknown about the HER2-low status across tumor types and changes in HER2 status between primary and metastatic samples. PATIENTS AND METHODS: HER2 expression by IHC was evaluated in 4701 patients with solid tumors. We have evaluated the HER2 expression by IHC and amplification by ISH in paired breast and gastric/gastroesophageal (GEJ) primary and metastatic samples. HER2 expression was correlated with ERBB2 genomic alterations evaluated by next-generation sequencing (NGS) in non-breast, non-gastric/GEJ samples. RESULTS: HER2 expression (HER2 IHC 1-3+) was found in half (49.8%) of the cancers, with HER2-low (1 or 2+) found in many tumor types: 47.1% in breast, 34.6% in gastric/GEJ, 50.0% in salivary gland, 46.9% in lung, 46.5% in endometrial, 46% in urothelial, and 45.5% of gallbladder cancers. The concordance evaluation of HER2 expression between primary and metastatic breast cancer samples showed that HER2 3+ remained unchanged in 87.1% with a strong agreement between primary and metastatic samples, with a weighted kappa (Κ) of 0.85 (95% confidence interval 0.79-0.91). ERBB2 alterations were identified in 117 (7.5%) patients with non-breast, non-gastric/GEJ solid tumors who had NGS testing. Of 1436 patients without ERBB2 alterations, 512 (35.7%) showed any level HER2 expression by IHC. CONCLUSION: Our results show that HER2-low expression is frequently found across tumor types. These findings suggest that many patients with HER2-low solid tumors might benefit from HER2-targeted therapies.

In vitro efficacy of ARQ 092, an allosteric AKT inhibitor, on primary fibroblast cells derived from patients with PIK3CA-related overgrowth spectrum (PROS).

Postzygotic mutations of the PIK3CA [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha] gene constitutively activate the PI3K/AKT/mTOR pathway in PIK3CA-related overgrowth spectrum (PROS) patients, causing congenital mosaic tissue overgrowth that even multiple surgeries cannot solve. mTOR inhibitors are empirically tested and given for compassionate use in these patients. PROS patients could be ideal candidates for enrolment in trials with PI3K/AKT pathway inhibitors, considering the "clean" cellular setting in which a unique driver, a PIK3CA mutation, is present. We aimed to assess the effects of blocking the upstream pathway of mTOR on PROS patient-derived cells by using ARQ 092, a potent, selective, allosteric, and experimental orally bioavailable and highly selective AKT-inhibitor with activity and long-term tolerability, currently under clinical development for treatment of cancer and Proteus syndrome. Cell samples (i.e., primary fibroblasts) were derived from cultured tissues obtained from six PROS patients [3 boys, 3 girls; aged 2 to 17 years] whose spectrum of PIK3A-related overgrowth included HHML [hemihyperplasia multiple lipomatosis; n = 1], CLOVES [congenital lipomatosis, overgrowth, vascular malformations, epidermal nevi, spinal/skeletal anomalies, scoliosis; n = 1], and MCAP [megalencephaly capillary malformation syndrome; n = 4]. We performed the following: (a) a deep sequencing assay of PI3K/AKT pathway genes in the six PROS patients' derived cells to identify the causative mutations and (b) a pathway analysis to assess the phosphorylation status of AKT [Ser473 and Thr308] and its downstream targets [pAKTS1 (Thr246), pRPS6 (Ser235/236), and pRPS6Kß1 (Ser371)]. The anti-proliferative effect of ARQ 092 was tested and compared to other PI3K/AKT/mTOR inhibitors [i.e., wortmannin, LY249002, and rapamycin] in the six PROS patient-derived cells. Using ARQ 092 to target AKT, a critical node connecting PI3K and mTOR pathways, we observed the following: (1) strong anti-proliferative activity [ARQ 092 at 0.5, 1, and 2.5 µM blunted phosphorylation of AKT and its downstream targets (in the presence or absence of serum) and inhibited proliferation after 72 h; rapamycin at 100 nM did not decrease AKT phosphorylation] and (2) less cytotoxicity as compared to rapamycin and wortmannin. We demonstrated the following: (a) that PROS cells are dependent on AKT; (b) the advantage of inhibiting the pathway immediately downstream of PI3K to circumventing problems depending on multiple classes a PI3K kinases; and (c) that PROS patients benefit from inhibition of AKT rather than mTOR. Clinical development of ARQ 092 in PROS patients is on going in these patients.

A Phase 1 study of ARQ 087, an oral pan-FGFR inhibitor in patients with advanced solid tumours.

BACKGROUND: ARQ 087 is an orally administered pan-FGFR inhibitor with multi-kinase activity. This Phase 1 study evaluated safety, pharmacokinetics, and pharmacodynamics of ARQ 087 and defined the recommended Phase 2 dose (RP2D). METHODS: Patients with advanced solid tumours received ARQ 087 administered initially at 25 mg every other day and dose-escalated from 25 to 425 mg daily (QD) continuous dosing. FGF19, 21, 23, and serum phosphate were assessed as potential biomarkers of target engagement. RESULTS: 80 patients were enrolled, 61 in dose-escalation/food-effect cohorts and 19 with pre-defined tumour types in the expansion cohort. The most common ARQ 087-related adverse events were fatigue (49%), nausea (46%), aspartate aminotransferase (AST) increase (30%), and diarrhoea (23%). Four patients (5%) experienced grade 1 treatment-related hyperphosphataemia. Dose-limiting toxicity was reversible grade 3 AST increase. The RP2D was 300 mg QD. Pharmacokinetics were linear and dose-proportional from 25 to 325 mg QD, and were unaffected by food. Statistically significant changes (P-value<0.05) suggest phosphate and FGF19 levels as markers of target engagement. In 18 evaluable patients with FGFR genetic alterations, 3 confirmed partial responses (two intrahepatic cholangiocarcinomas (iCCA) with FGFR2 fusions and one urothelial cancer with FGFR2 and FGF19 amplification) and two durable stable disease at ⩾16 weeks with tumour reduction (FGFR2 fusion-positive iCCA and adrenocortical carcinoma with FGFR1 amplification) were observed. CONCLUSIONS: ARQ 087 had manageable toxicity at the RP2D of 300 mg QD, showed pharmacodynamics effects, and achieved objective responses, notably in patients with FGFR2 genetic alterations.

Systemic effects of locally injected platelet rich plasma in a rat model: an analysis on muscle and bloodstream.

Abundant evidence suggests that growth factors, contained in platelets alpha granules, may play a key role in the early stages of the muscle healing process with particular regard to the inflammatory phase. Although the contents of the platelet-rich plasma preparations have been extensively studied, the biological mechanisms involved as well as the systemic effects and the related potential doping implications of this approach are still largely unknown. The aim of the present study was to investigate whether local platelet-rich plasma administration may modify the levels of specific cytokines and growth factors both in treated muscle and bloodstream in rats. An additional aim was to investigate more deeply whether the local platelet-rich plasma administration may exert systemic effects by analyzing contralateral lesioned but untreated muscles. The results showed that platelet-rich plasma treatment induced a modification of certain cytokines and growth factor levels in muscle but not in the bloodstream, suggesting that local platelet-rich plasma treatment influenced directly or, more plausibly, indirectly the synthesis or recruitment of cytokines and growth factors at the site of injury. Moreover, the observed modifications of cytokine and growth factor levels in contralateral injured but not treated muscles, strongly suggested a systemic effect of locally injected platelet-rich plasma.

A phase I dose escalation study of oral c-MET inhibitor tivantinib (ARQ 197) in combination with gemcitabine in patients with solid tumors.

BACKGROUND: Tivantinib (ARQ 197) is an orally available, non-adenosine triphosphate competitive, selective c-MET inhibitor. The primary objective of this study was to evaluate the safety, tolerability and to establish the recommended phase II dose (RP2D) of tivantinib and gemcitabine combination. PATIENTS AND METHODS: Patients with advanced or metastatic solid tumors were treated with escalating doses of tivantinib (120-360 mg capsules) in combination with gemcitabine (1000 mg/m(2) weekly for 3 of 4 weeks). Different schedules of administration were tested and modified based on emerging preclinical data. Tivantinib was given continuously, twice a day (b.i.d.) for 2, 3 or 4 weeks of a 28-day cycle or on a 5-day on, 2-day off schedule (the day before and day of gemcitabine administration). RESULTS: Twenty-nine patients were treated with gemcitabine and escalating doses of tivantinib: 120 mg b.i.d. (n = 4), 240 mg b.i.d. (n = 6) and 360 mg b.i.d. (n = 19). No dose-limiting toxicities were observed in escalation. The RP2D was 360 mg b.i.d. daily, and 45 additional patients were enrolled in the expansion cohort. Grade ≥3 treatment-related toxicities were observed in 54 of 74 (73%) patients with the most common being neutropenia (43%), anemia (30%), thrombocytopenia (28%) and fatigue (15%). There was one treatment-related death due to neutropenia. Administration of gemcitabine did not affect tivantinib concentration. Fifty-six patients were assessable for response. Eleven (20%) patients achieved a partial response and 26 (46%) had stable disease (SD), including 15 (27%) who achieved SD for over 4 months. Ten of 37 patients with clinical benefit had prior exposure to gemcitabine. CONCLUSION: The combination of tivantinib at its monotherapy dose and standard dose gemcitabine was safe and tolerable. Early signs of antitumor activity may warrant further development of this combination in nonsmall-cell lung cancer, ovarian, pancreatic and cholangiocarcinoma. CLINICALTRIALSGOV IDENTIFIER: NCT00874042.

A Phase-1b study of tivantinib (ARQ 197) in adult patients with hepatocellular carcinoma and cirrhosis.

BACKGROUND: The mesenchymal-epithelial transition factor (MET) receptor is dysregulated in hepatocellular carcinoma (HCC), and tivantinib (ARQ 197) is an oral, selective, MET inhibitor. METHODS: This Phase-1b study assessed tivantinib safety as primary objective in patients with previously treated HCC and Child-Pugh A or B liver cirrhosis. Patients received oral tivantinib 360 mg twice daily until disease progression or unacceptable toxicity. RESULTS: Among 21 HCC patients, common drug-related adverse events (AEs) were neutropaenia, anaemia, asthenia, leucopaenia, anorexia, diarrhoea, and fatigue. No drug-related worsening of liver function or performance status occurred, but one Child-Pugh B patient experienced drug-related bilirubin increase. Four patients had drug-related serious AEs, including one neutropaenia-related death. Haematologic toxicities were more frequent than in previous tivantinib studies but were manageable with prompt therapy. Best response was stable disease (median, 5.3 months) in 9 of 16 evaluable patients (56%). Median time to progression was 3.3 months. CONCLUSION: Tivantinib demonstrated a manageable safety profile and preliminary antitumour activity in patients with HCC and Child-Pugh A or B cirrhosis.

Sexual dysfunction in multiple sclerosis: The impact of different MSISQ-19 cut-offs on prevalence and associated risk factors.

BACKGROUND: Although multiple sclerosis (MS) Intimacy and Sexuality Questionnaire-19 (MSISQ-19) is a widely applied tool, no unique definition of sexual dysfunction (SD) based on its score exists. OBJECTIVE: To explore the impact of different MSISQ-19 cut-offs on SD prevalence and associated risk factors, providing relevant information for its application in research and clinical settings. METHODS: After defining SD according to two different MSISQ-19 cut-offs in 1155 people with MS (pwMS), we evaluated SD prevalence and association with sociodemographic and clinical features, mood status and disability via logistic regression. RESULTS: Depending on the chosen cut-off, 45% to 54% of pwMS reported SD. SD defined as MSISQ-19 score >30 was predicted by age (OR=1.01, p=0.047), cognition (OR=0.96, p=0.004) and anxiety (OR=1.03, p=0.019). SD defined as a score >3 on any MSISQ-19 item was predicted by motor disability (OR=1.12, p=0.003) and cognition (OR= 0.96, p=0.002). CONCLUSION: Applying different MSISQ-19 cut-offs influences both the estimated prevalence and the identification of risk factors for SD, a finding that should be considered during study planning and data interpretation. Preserved cognition exerts a protective effect towards SD regardless from the specific study setting, representing a key point for the implementation of preventive and therapeutic strategies.

Steroid resistance in nasal polyposis: role of glucocorticoid receptor and TGF-beta1.

BACKGROUND: Glucocorticoids (GCs) are considered drugs of choice for treating nasal polyps (NPs). However, a subset of patients shows a limited clinical response even to high doses of GCs. Altered expression of glucocorticoid receptors (GRs), namely GR-alpha; and GR-beta;, is a potential mechanism underlying GC insensitivity. GCs modulate the expression of several cytokines, including transforming growth factor-beta (TGF-beta), which may contribute to cellular proliferation in NPs. The study investigates some biomolecular features of GC-resistant NPs, and examines possible differences from normal mucosa (NM). METHODOLOGY: Radioligand binding assay (binding) was used to determine GR-alpha; binding capacity; Western blotting was used to evaluate GR-alpha;, GR-beta;, and TGF-beta; expression and GR-alpha; subcellular distribution. NPs were sampled in 32 patients during ethmoidectomy; NM was taken from 15 healthy patients during rhinoplasty. RESULTS: GR-alpha; was present in NPs and NM, with lower affinity for the ligand in NPs. GR-alpha; was prevalent in the cytosol of NPs that were GR-alpha-negative to the binding assay. GR-beta was expressed in NPs and absent in the majority of NM. TGF-beta1 expression was higher in NPs than in NM. CONCLUSIONS: GR-beta and TGF-beta1 might be involved in NP pathogenesis, but their role in modulating GC sensitivity is still unclear.

Digital therapeutics in neurology.

Digital therapeutics (DTx) is a section of digital health defined by the DTx Alliance as "delivering evidence-based therapeutic interventions to patients that are driven by software to prevent, manage, or treat a medical disorder or disease. They are used independently or in concert with medications, devices, or other therapies to optimize patient care and health outcomes". Chronic disabling diseases could greatly benefit from DTx. In this narrative review, we provide an overview of DTx in the care of patients with neurological dysfunctions.

Isatuximab plus atezolizumab in patients with advanced solid tumors: results from a phase I/II, open-label, multicenter study.

BACKGROUND: The anti-CD38 antibody isatuximab is approved for the treatment of relapsed/refractory multiple myeloma, but there are no data on its efficacy in solid tumors. This phase I/II study (NCT03637764) assessed the safety and activity of isatuximab plus atezolizumab (Isa + Atezo), an anti-programmed death-ligand 1 (PD-L1) antibody, in patients with immunotherapy-naive solid tumors: epithelial ovarian cancer (EOC), glioblastoma (GBM), hepatocellular carcinoma (HCC), and squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Phase I assessed safety, tolerability, pharmacokinetics, pharmacodynamics, and the recommended phase II dose (RP2D) of isatuximab 10 mg/kg intravenously (i.v.) every week for 3 weeks followed by once every 3 weeks + atezolizumab 1200 mg i.v. every 3 weeks. Phase II used a Simon's two-stage design to assess the overall response rate or progression-free survival rate at 6 months (GBM cohort). Interim analysis was carried out at 6 months following first dose of the last enrolled patient in each cohort. Pharmacodynamic biomarkers were tested for CD38, PD-L1, tumor-infiltrating immune cells, and FOXP3+ regulatory T cells (Tregs) in the tumor microenvironment (TME). RESULTS: Overall, 107 patients were treated (EOC, n = 18; GBM, n = 33; HCC, n = 27; SCCHN, n = 29). In phase I, Isa + Atezo showed an acceptable safety profile, no dose-limiting toxicities were observed, and RP2D was confirmed. Most patients experienced ≥1 treatment-emergent adverse event (TEAE), with ≤48.5% being grade ≥3. The most frequent TEAE was infusion reactions. The study did not continue to stage 2 based on prespecified targets. Tumor-infiltrating CD38+ immune cells were reduced and almost cleared after treatment. Isa + Atezo did not significantly modulate Tregs or PD-L1 expression in the TME. CONCLUSIONS: Isa + Atezo had acceptable safety and tolerability. Clinical pharmacodynamic evaluation revealed efficient target engagement of isatuximab via treatment-mediated reduction of CD38+ immune cells in the TME. Based on clinical data, CD38 inhibition does not improve responsiveness to PD-L1 blockade in these patients.

Ischemia/reperfusion injury is increased and cardioprotection by a postconditioning protocol is lost as cardiac hypertrophy develops in nandrolone treated rats.

We hypothesized that nandrolone (ND)-abuse induces cardiac hypertrophy, increases myocardial susceptibility to ischemia/reperfusion (I/R) injury, and reduces responsiveness to postconditioning (PostC) cardioprotection. Wistar-rats were ND treated for 2 weeks (short_ND) or 10 weeks (long_ND). Vehicle-treated rats served as controls. Hearts were retrogradely perfused and left ventricular pressure (LVP) was measured before and after 30-min global ischemia. In subgroups of hearts, to induce cardioprotection a PostC protocol (five cycles of 10-s reperfusion and 10-s ischemia) was performed. ß-adrenoreceptors, kinases (Akt and GSK-3ß) and phosphatases (PP2A sub A and PP2A sub B) were examined by Western blot before and after ischemia. After 120-min reperfusion, infarct size was measured. Short_ND slightly increased cardiac/body weight ratio, but did not affect cardiac baseline nor post-ischemic contractile function or infarct size when compared to vehicle hearts. However, PostC limited cardiac dysfunction much more in short_ND hearts than the other groups. Although cardiac/body weight ratio markedly increased after long_ND, baseline LVP was not affected. Yet, post-ischemic contracture and infarct size were exacerbated and PostC was unable to reduce infarct size and ventricular dysfunction. While short_ND increased phosphatases, non-phosphorylated and phosphorylated Akt, long_ND reduced phosphatase-expression and Akt phosphorylation. Both short_ND and long_ND had no effect on the GSK-3ß-phosphorylation but increased the expression of ß(2)-adrenoreceptors. In reperfusion, PostC increased Akt phosphorylation regardless of protective effects, but reduced phosphatase-expression in protected hearts only. In conclusion, short_ND improves post-ischemic myocardial performance in postconditioned hearts. However, long_ND increases myocardial susceptibility to I/R injury and abolishes cardioprotection by PostC. This increased susceptibility might be related to steroid-induced hypertrophy and/or to altered enzyme expression/phosphorylation.

BMI influences CD20 kinetics in multiple sclerosis patients treated with ocrelizumab.

OBJECTIVES: Ocrelizumab (OCR) is a humanized monoclonal antibody targeting CD20 antigen exposed on B cells surface. Kinetic of B-cells repopulation after depletion therapy shows high intra and inter-individual variability. The aim of this study was to explore the influence of Body Mass Index (BMI) on kinetic of B-cell repopulation after treatment with OCR and on treatment response. METHODS: 108 Multiple Sclerosis (MS) patients were enrolled at the time of the first dose of OCR administration and prospectively evaluated. Clinical, instrumental activity and disability progression were analyzed. According to B cells count, patients were divided into two groups: with fast (FR) and with slow (SR) repopulation rate, respectively. RESULTS: Significant reduction of disease activity was observed in all patients and a stabilization of disease was obtained in progressive patients. Patients with FR had higher BMI compared to patients with a SR (p<0.001). Contrariwise no correlation between repopulation rate and treatment effectiveness was disclosed. CONCLUSIONS: In a real world setting we confirmed the effectiveness of OCR in relapsing remitting and progressive patients; patients with higher BMI had a FR. This suggests considering BMI for administration schedule although further investigations with longer follow up could improve treatment protocol and patient selection.

A small molecule based on the pRb2/p130 spacer domain leads to inhibition of cdk2 activity, cell cycle arrest and tumor growth reduction in vivo.

One strategy in the development of anticancer therapeutics has been to arrest malignant proliferation through inhibition of the enzymatic activity of cyclin-dependent kinases (cdks), which are key regulatory molecules of the cell cycle. Over the past few years, numerous compounds with remarkable cdk inhibitory activity have been studied in cancer therapy, although it is very difficult to point out the best cdk to target. An excellent candidate appears to be cdk2, whose alteration is a pathogenic hallmark of tumorigenesis. The small molecule described in our study showed an inhibitory effect on the kinase activity of cdk2, a significant growth arrest observed in a colony formation assay and a reduction in the size of the tumor in nude mice, thus suggesting its potential role as a promising new type of mechanism-based antitumor drug, also for the treatment of hyperproliferative disorders.

Fake news, influencers and health-related professional participation on the Web: A pilot study on a social-network of people with Multiple Sclerosis.

BACKGROUND: Over the last few decades, patients have increasingly been searching for health information on the Internet. This aspect of information seeking is important, especially for people affected by chronic pathologies and require lifelong treatment and management. These people are usually very well informed about the disease but are nonetheless vulnerable to hopes of being cured or saved, often amplified by misinformation, myths, legends, and therapies that are not always scientifically proven. Many studies suggest that some individuals prefer to rely on the Internet as their main source of information, often hindering the patient-doctor relationship. A professional approach is imperative to maintain confidentiality, honesty, and trust in the medical profession. OBJECTIVE: we aimed to examine, in a medically supervised Italian web community (SMsocialnetwotk.com) dedicated to people with Multiple Sclerosis (pwMS), the posts shared by users and to verify the reliability of contents of posts shared by users pinpointed as Influencers through an online questionnaire. METHODS: we grouped the posts published on SMsocialnetwork from April to June 2015 into those with medical content (scientifically correct or fake news), and those related to social interactions. Later, we gave a questionnaire to the community asking to identify the three users/Influencers providing the most reliable advice for everyday life with MS and the three users/Influencers providing the most useful information about MS treatments. RESULTS: 308 posts reported scientific and relevant medical information, whereas 72 posts included pieces of fake news. 1420 posts were of general interest. Four out of the 6 Influencers had written only posts with correct medical information (3 were pwMS, 1 was a Neurologist) and never any fake news. The remaining 2 appointed Influencers (2 pwMS) had written only posts about general interests. CONCLUSION: the identification of fake news and their authors has shown that the latter are never appointed as Influencers. SMsocialnetwork.com acted as a "web safe environment" where the Influencers contributed by sharing only correct medical information and never fake news. We speculate that the presence of neurologists and psychologists supervising the information flow might have contributed to reduce the risk of fake news spreading and to avoid their acquisition of authoritative meaning.

Viral infections as a cause of cancer (review).

In order to promote carcinogenesis multiple factors must be orchestrated. The alteration of the cellular genome after a carcinogenic exposure may result in malignancy if apoptosis is prevented and the immune surveillance fails to eliminate the transformed cell. Infectious agents may exert these properties and transform a host cell. Viruses associated with human cancer are known as 'tumor viruses'. Most of them are capable of integrating into the host genome and have the ability to immortalize the target cell in order to allow their own replication. The infected cell expresses the viral genes, which are able to induce cell growth, proliferation and prevent apoptosis. This review focuses on Epstein-Barr virus, human papilloma virus, hepatitis C virus, hepatitis B virus, human herpes virus 8 and human T-cell leukemia virus, since they have been already established as causative agents of human cancer. An understanding of the viral replication mechanism may provide new targets for the development of specified viral therapy that may have an impact not only on viral infections but in human cancer as well.

Topical kanamycin: an effective therapeutic option in aerobic vaginitis.

Eighty-one patients with clinical diagnosis of aerobic vaginitis (AV) were included in the study. The patients were randomized for treatment, 45 with kanamycin (100 mg vaginal ovules for 6 days, consecutively) and 36 with meclocycline (35 mg vaginal ovules for 6 days, consecutively). The patients were examined before starting the study, 1-2 days after treatment and 30 days after the end of the study. At the first follow-up the patients showed different levels of symptom reduction. Reduction in the presence of leukocytes, vaginal mucosa burning and itching were statistically significant in the group treated with kanamycin with respect to the group treated with meclocycline. Moreover, there was also reduced isolation of Enterobacteriaeae (97%) in the group treated with kanamycin versus those treated with meclocycline (76%). At the second follow-up, vaginal homeostasis (normalization of pH and presence of lactobacilli) was more evident in the kanamycin-treated group. In conclusion, our data suggest that the topical use of kanamycin could be considered a specific antibiotic for the therapy of this new pathology.

Rapid determination of anti-estrogens by gas chromatography/mass spectrometry in urine: Method validation and application to real samples.

A fast screening protocol was developed for the simultaneous determination of nine anti-estrogenic agents (aminoglutethimide, anastrozole, clomiphene, drostanolone, formestane, letrozole, mesterolone, tamoxifen, testolactone) plus five of their metabolites in human urine. After an enzymatic hydrolysis, these compounds can be extracted simultaneously from urine with a simple liquid-liquid extraction at alkaline conditions. The analytes were subsequently analyzed by fast-gas chromatography/mass spectrometry (fast-GC/MS) after derivatization. The use of a short column, high-flow carrier gas velocity and fast temperature ramping produced an efficient separation of all analytes in about 4 min, allowing a processing rate of 10 samples/h. The present analytical method was validated according to UNI EN ISO/IEC 17025 guidelines for qualitative methods. The range of investigated parameters included the limit of detection, selectivity, linearity, repeatability, robustness and extraction efficiency. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. Therefore, the performances of the method are comparable to the ones obtainable from traditional GC/MS analysis. The method was successfully tested on real samples arising from clinical treatments of hospitalized patients and could profitably be used for clinical studies on anti-estrogenic drug administration.