D5S2500 is an ambiguously characterized STR: Identification and description of forensic microsatellites in the genomics age.

In the process of establishing short tandem repeat (STR) sequence variant nomenclature guidelines in anticipation of expanded forensic multiplexes for massively parallel sequencing (MPS), it was discovered that the STR D5S2500 has multiple positions and genomic characteristics reported. This ambiguity is because the marker named D5S2500 consists of two different microsatellites forming separate components in the capillary electrophoresis multiplexes of Qiagen's HDplex (Hilden, Germany) and AGCU ScienTech's non-CODIS STR 21plex (Wuxi, Jiangsu, China). This study outlines the genomic details used to identify each microsatellite and reveals the D5S2500 marker in HDplex has the correctly assigned STR name, while the D5S2500 marker in the AGCU 21plex, closely positioned a further 1643 nucleotides in the human reference sequence, is an unnamed microsatellite. The fact that the D5S2500 marker has existed as two distinct STR loci undetected for almost ten years, even with reported discordant genotypes for the standard control DNA, underlines the need for careful scrutiny of the genomic properties of forensic STRs, as they become adapted for sequence analysis with MPS systems. We make the recommendation that precise chromosome location data must be reported for any forensic marker under development but not in common use, so that the genomic characteristics of the locus are validated to the same level of accuracy as its allelic variation and forensic performance. To clearly differentiate each microsatellite, we propose the name D5S2800 be used to identify the Chromosome-5 STR in the AGCU 21plex.

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

Revision of the SNPforID 34-plex forensic ancestry test: Assay enhancements, standard reference sample genotypes and extended population studies.

A revision of an established 34 SNP forensic ancestry test has been made by swapping the under-performing rs727811 component SNP with the highly informative rs3827760 that shows a near-fixed East Asian specific allele. We collated SNP variability data for the revised SNP set in 66 reference populations from 1000 Genomes and HGDP-CEPH panels and used this as reference data to analyse four U.S. populations showing a range of admixture patterns. The U.S. Hispanics sample in particular displayed heterogeneous values of co-ancestry between European, Native American and African contributors, likely to reflect in part, the way this disparate group is defined using cultural as well as population genetic parameters. The genotyping of over 700 U.S. population samples also provided the opportunity to thoroughly gauge peak mobility variation and peak height ratios observed from routine use of the single base extension chemistry of the 34-plex test. Finally, the genotyping of the widely used DNA profiling Standard Reference Material samples plus other control DNAs completes the audit of the 34-plex assay to allow forensic practitioners to apply this test more readily in their own laboratories.

Forensic performance of two insertion-deletion marker assays.

Improving the amplification and analysis of highly degraded DNA extracts has been a longstanding area of research in forensic genetics. One of the most promising recent developments in analysis of degraded DNA is the availability of short, biallelic insertion-deletion length polymorphisms (InDels) in highly multiplexed assays. InDels share many of the favourable characteristics of single-nucleotide polymorphisms (SNPs) that make them ideal markers for analysis of degraded DNA, including: analysis in short amplicon size ranges, high multiplexing capability and low mutation rates. In addition, as length-based polymorphisms, InDels can be analysed with the same simple dye-labelled PCR primer methods as standard forensic short tandem repeats. Separation and detection of fluorescently dye-labelled PCR products by capillary electrophoresis eliminate the multiple step protocols required by SNP typing with single-base extension assays and provide a closer relationship between the input DNA and the profile peak height ratios. Therefore InDel genotyping represents an effective new approach for human identification that adds informative new loci to the existing battery of forensic markers. To assess the utility of InDels for forensic analysis, we characterised population variation with two InDel identification assays: the 30-plex Qiagen DIPplex panel and a 38-plex panel developed by Pereira et al. in 2009. Allele frequencies were generated for the 68 markers in US African American, Caucasian, East Asian and Hispanic samples. We made a thorough assessment of the individual and combined performance of the InDel sets, as well as characterising profile artifacts and other issues related to the routine use of these newly developed forensic assays based on artificially degraded DNA and mixed source samples.

Source and clinical motivation of orders for contrast-enhanced sonography (CEUS) of the liver: A prospective single-center survey.

Contrast-enhanced sonography (CEUS) has become a routine part of diagnostic imaging of the liver. Its possibilities, limitations, and indications have been defined in adequately large clinical series and in guidelines and recommendations. We prospectively evaluated physicians' orders for hepatic CEUS received in the radiology department of a large oncology center in Naples, Italy from May 2009 to April 2010. Radiologists performing the CEUS examinations filled out a form that included patient demography, source and type of patient referral, and clinical indications for the examination. During the study period, 564 patients aged 17-86 years (mean, 58 years) were referred to our department for CEUS liver studies (total: 644; 491 outpatient studies, 153 inpatient studies). This included 4 examinations that were ordered by the patient's physician but not performed by our staff. The majority of the CEUS examinations (n = 583; 90.5%) were regularly scheduled procedures ordered by clinical specialists from our center (77.3%) or other centers (11.8%); by general practitioners (on their own initiative) (0.8%); or by other figures (0.6%). The remaining 61 examinations (9.5%) were unscheduled procedures done on the initiative of a radiologist following conventional sonography (US). Fewer than half (47.8%) of the examinations were requested as first-line assessments. The others were ordered to clarify inconclusive findings generated by conventional US (30%) or by a more sophisticated imaging study (CT, MRI, PET) (16.1%) or to resolve discrepancies between CT, MRI, and/or PET findings (6%). CEUS is a relatively noninvasive, low-cost imaging study that is simple to perform and requires no particular patient preparation. This may explain its increasing use to clarify doubts raised by conventional US and other more sophisticated imaging studies.

Autosomal SNP typing of forensic samples with the GenPlex™ HID System: results of a collaborative study.

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

Forensic application of the Affymetrix human mitochondrial resequencing array.

In the field of forensic DNA testing, sequencing regions of the mitochondrial genome is performed when insufficient genomic DNA is present for traditional autosomal short tandem repeat (STR) testing. Sequencing coding region polymorphisms in the mitochondrial genome can be useful for resolving individuals who have the identical HV1 and HV2 control region sequence. Various methods and strategies have been established to interrogate coding region polymorphisms. These range from SNP assays probing sites most likely to differentiate individuals based on their HV1/HV2 sequence to the use of mass spectrometry to pyrosequencing. Here we evaluate the potential of the Affymetrix GeneChip Mitochondrial Resequencing Array (version 2.0) for forensic applications.

The impact of additional Y-STR loci on resolving common haplotypes and closely related individuals.

Commercial Y-STR kits have permitted laboratories to go beyond the original nine minimal haplotype loci (MHL) and to discover the advantage of additional Y-STR loci in resolving common haplotypes. In an effort to examine the impact of Y-STR markers beyond the 17 loci now available in commercial kit form, new Y-STR loci are being investigated on a common set of samples representative of the major U.S. population groups. Additional Y-STRs can also increase the power of discrimination between closely related male individuals, which is important not only in forensics but also in the paternity and genetic genealogy communities.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Radiofrequency ablation in patients with primary breast carcinoma: a pilot study in 26 patients.

BACKGROUND: The authors performed a pilot trial of ultrasound-guided percutaneous radiofrequency ablation (RFA) in patients with T1 and T2 breast tumors 1) to confirm complete coagulative necrosis of tumor tissue and 2) to determine the safety and complications related to this treatment. METHODS: Twenty-six patients with biopsy-proven, invasive breast carcinoma underwent RFA of their breast tumors followed by immediate resection. Treatment was planned to ablate the tumor and a 5 mm margin of surrounding breast tissue. Tumor viability after RFA was assessed by hematoxylin and eosin and nicotinamide adenine dinucleotide vital staining. RESULTS: Twenty patients (77%) had T1 tumors, and six patients (23%) had T2 tumors. The mean greatest dimension of tumors that were treated with RFA was 1.8 cm (range, 0.7-3.0 cm). The mean treatment time for two-phase RFA treatment was 15 minutes and 23 seconds (range, from 6 minutes and 25 seconds to 24 minutes and 54 seconds). Coagulation necrosis of the tumor was complete in 25 of 26 patients (96%): One patient had a microscopic focus of viable tissue adjacent to the needle shaft site. A single patient (1 of 26 patients; 4%) had a complication related to RFA: a full thickness burn of the skin overlying a tumor that was immediately beneath the skin. CONCLUSIONS: This pilot experience with RFA in the treatment of patients with early-stage, primary breast carcinoma revealed that 1) coagulative necrosis of the entire tumor occurred in 96% of the patients, and 2) the treatment was safe, with only a 4% complication rate. The authors have initiated a trial of RFA alone (no resection) for patients with T1 and T2 breast tumors that will include sentinel lymph node mapping and postablation irradiation.

Calculating sequence-dependent melting stability of duplex DNA oligomers and multiplex sequence analysis by graphs.

The analytical methods for characterizing DNA sequence-dependent thermodynamic stability have been reviewed. A set of n-n sequence stability parameters is presented. Examples in which these values are used to calculate the thermodynamic stability of short duplex DNA oligomers are presented. The problem of determining sets of isothermal sequences is addressed by representing DNA sequences as graphs. Representing DNA sequences by a graph descriptor with special mathematical properties minimizes the computational difficulty of determining the number of DNA sequences with identical predicted thermodynamic stability. This is achieved by replacement of a whole set of sequences by a single representative. Applications of this concept were demonstrated for sequences assembled from individual bases and sequences assembled from oligomeric blocks.

Quality control of PCR primers used in multiplex STR amplification reactions.

Reliable amplification of short tandem repeat (STR) DNA markers with the polymerase chain reaction (PCR) is dependent on high quality PCR primers. The particular primer combinations and concentrations are especially important with multiplex amplification reactions where multiple STR loci are simultaneously copied. Commercially available kits are now widely used for STR amplification and subsequent DNA typing. We present here the use of high performance liquid chromatography (HPLC) and time-of-flight mass spectrometry (TOF-MS) methods for characterization of commercially available STR kits.

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions.

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.

Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography.

Currently, a major focus of human genetics is the utilization of single-nucleotide polymorphisms for clinical diagnostics, whole-genome linkage disequilibrium screens to identify common disease genes such as Alzheimer disease, determination of the recent evolutionary history of a species, and the process of speciation. We have examined single-nucleotide extension coupled with high-performance liquid chromatography as a method to simultaneously genotype two SNPs occurring in the coding region of the HFE gene that produce clinical effects. This assay allows concurrent genotyping of the C282Y and H63D mutations in 11 min and is 100% concordant with current testing methods for both of these mutations.

Levovist-enhanced Doppler sonography versus spiral computed tomography to evaluate response to percutaneous ethanol injection in hepatocellular carcinoma.

The aim of the current study was to compare Levovist-enhanced power Doppler (PD) imaging with contrast-enhanced spiral computed tomography (CT) in the evaluation of intratumoral vascularity of hepatocellular carcinomas at diagnosis and after percutaneous ethanol injection (PEI). Nineteen patients with hepatocellular carcinoma (HCC) underwent PD with and without Levovist and spiral CT at diagnosis and 1 month after PEI treatment. Compared to spiral CT at baseline evaluation, the PD showed intratumoral vascularity in 36.8% of the cases; this percentage reached 78.9% after Levovist enhancement. One month after PEI, only 5 out of 19 treated HCCs appeared as hypodense areas at CT and showed no contrast enhancement. Only 3 of the 14 patients with a positive spiral CT scan were positive at the PD performed without the Levovist administration (sensitivity, 21.4%). The use of contrast-enhanced ultrasonography led to detection of residual signal in six other HCCs treated by ethanol injection (sensitivity, 64.2%). We confirm that spiral CT is the most sensitive and accurate technique in evaluating the effect of ethanol injection in HCC. It correctly identifies most cases of treatment failure as enhanced areas within the lesion. The lower rate of detection of tumoral vascularity by Doppler sonography was significantly increased by Levovist. The evidence of residual vascularity within HCC at Levovist Doppler sonography allows the targeting of additional ethanol injections.

Radiofrequency ablation of hepatocellular cancer in 110 patients with cirrhosis.

OBJECTIVE: To determine the treatment efficacy, safety, local tumor control, and complications related to radiofrequency ablation (RFA) in patients with cirrhosis and unresectable hepatocellular carcinoma (HCC). SUMMARY BACKGROUND DATA: Most patients with HCC are not candidates for resection because of tumor size, location, or hepatic dysfunction related to cirrhosis. RFA is a technique that permits in situ destruction of tumors by means of local tissue heating. METHODS: One hundred ten patients with cirrhosis and HCC (Child class A, 50; B, 31; C, 29) were treated during a prospective study using RFA. Patients were treated with RFA using an open laparotomy, laparoscopic, or percutaneous approach with ultrasound guidance to place the RF needle electrode into the hepatic tumors. All patients were followed up at regular intervals to detect treatment-related complications or recurrence of disease. RESULTS: All 110 patients were followed up for at least 12 months after RFA (median follow-up 19 months). Percutaneous or intraoperative RFA was performed in 76 (69%) and 34 patients (31%), respectively. A total of 149 discrete HCC tumor nodules were treated with RFA. The median diameter of tumors treated percutaneously (2.8 cm) was smaller than that of lesions treated during laparotomy (4.6 cm). Local tumor recurrence at the RFA site developed in four patients (3.6%); recurrent HCC subsequently developed in other areas of the liver in all four. New liver tumors or extrahepatic metastases developed in 50 patients (45. 5%), but 56 patients (50.9%) had no evidence of recurrence. There were no treatment-related deaths, but complications developed in 14 patients (12.7%) after RFA. CONCLUSIONS: In patients with cirrhosis and HCC, RFA produces effective local control of disease in a significant proportion of patients and can be performed safely with minimal complications.

Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides.

Effects of different end sequences on melting, circular dichroism spectra (CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both sides by sequences differing in AT content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from a van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming that the melting transition is two-state. Melting free energies (20 degrees C) evaluated from DSC melting experiments on the four duplex DNAs ranged from -52.2 to -77.5 kcal/mol. Free energies based on the van't Hoff analysis were -37.9 to -58.8 kcal/mol. Although the values are different, trends in the melting free energies of the four duplex DNAs as a function of sequence were identical in both DSC and optical analyses. Subject to several assumptions, values for the initiation free energy were estimated for each duplex, defined as DeltaG(int) = DeltaG(cal) - DeltaG(pred), where DeltaG(cal) is the experimental free energy at 20 degrees C determined from the experimentially measured values of the transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal). The predicted free energy of the sequence, DeltaG(pred)(20 degrees C), is obtained using published nearest-neighbor sequence stability values. For three of the four duplexes, values of DeltaG(int) are essentially nil. In contrast, the duplex with 81.8% GC has a considerably higher estimate of DeltaG(int) = 7.1 kcal/mol. The CD spectra for the six duplexes collected over the wavelength range from 200 to 320 nm are also sequence-dependent. Factor analysis of the CD spectra by singular value decomposition revealed that the experimental CD spectra could be reconstructed from linear combinations of two minor and one major subspectra. Changes in the coefficients of the major subspectrum for different sequences reflect incremental sequence-dependent variations of the CD spectra. Equilibrium binding by BamHI restriction endonuclease to the 40 base pair DNAs whose central eight base pairs contain the recognition sequence for BamHI restriction enzyme bounded by A.T base pairs, 5'-A-GGATCC-A-3' was investigated. Binding assays were performed by titering BamHI against a constant concentration of each of the duplex DNA substrates, in the absence of Mg(2+), followed by analysis by gel retardation. Under the conditions employed, the enzyme binds but does not cleave the DNAs. Results of the assays revealed two binding modes with retarded gel mobilities. Binding isotherms for the fraction of bound DNA species versus enzyme concentration for each binding mode were constructed and analyzed with a simple two-step equilibrium binding model. This analysis provided semiquantitative estimates on the equilibrium binding constants for BamHI to the four DNAs. Values obtained for the binding constants varied only 7-fold and ranged from 6 x 10(-)(8) to 42 x 10(-)(8) M, with binding free energies from -8.6 to -9.7 (+/- 0.2) kcal/mol depending on the sequence that flanks the enzyme binding site. Unlike what was found earlier in binding studies of the 22 base pair duplexes that constitute the core modules of the present 40-mers [Riccelli, P. V., Vallone, P. M., Kashin, I., Faldasz, B. D., Lane, M. J., and Benight, A. S. (1999) Biochemistry 38, 11197-11208], no obvious relationship between binding and stability was found for these longer DNAs. Apparently, effects of flanking sequence stability on restriction enzyme binding may only be measurable in very short duplex deoxyoligonucl

Weekly docetaxel plus gemcitabine or vinorelbine in refractory advanced breast cancer patients: a parallel dose-finding study. Southern Italy Cooperative Oncology Group (SICOG).

PURPOSE: The objective of this study was to determine the docetaxel MTD when combined with gemcitabine or vinorelbine in advanced breast cancer patients who had received previous anthracycline-based chemotherapy for advanced disease. PATIENTS AND METHODS: Advanced breast cancer patients aged between 18 and 70 with ECOG PS 0-2 who had not responded to, or had relapsed after, first-line anthracycline-based chemotherapy, were randomized to receive either gemcitabine 1000 mg/m2 or vinorelbine 25 mg/m2 in combination with escalating doses of docetaxel (starting from 30 mg/m2), all on days 1 and 8 every three weeks. Escalation was stopped if > 33% of patients treated at a given dose level showed DLT at the first cycle. RESULTS: A total of 34 patients with locally advanced (8) or metastatic disease (26) were treated, for a total of 94 cycles delivered. Nineteen patients received docetaxel in combination with gemcitabine and 15 with vinorelbine. All patients had been pretreated with anthracyclines, and 24 of 34 had also received weekly dose-dense paclitaxel. A docetaxel dose of 40/m2 proved to be safe when combined on days 1 and 8 with gemcitabine, while a dose of 35 mg/m2 was tolerated in combination with vinorelbine. Overall, nine episodes of DLT, all of them neutropenia, occurred at the first cycle. Considering all 94 cycles, grades 3 or 4 neutropenia and thrombocytopenia occurred in 15 (44%), and 7 (20%) patients. Non-hematologic toxicity was mild, except for three cases of grade 2 peripheral neuropathy. All patients were assessed for response on an 'intent-to-treat' basis. Overall, five partial responses were recorded (docetaxel + gemcitabine = 3 and docetaxel + vinorelbine = 2), for a 15% (95% CI: 5%-31%) overall response rate. Only 1 of 24 (4%) patients who had received weekly dose-dense paclitaxel responded to treatment. CONCLUSIONS: The weekly docetaxel administration in combination with either gemcitabine or vinorelbine is a well-tolerated treatment for heavily pretreated advanced breast cancer patients. This approach, although sometimes capable of achieving a major response, does not seem advisable in advanced breast cancer patients refractory to both anthracyclines and paclitaxel.

Phase I clinical trial of liposomal daunorubicin in hepatocellular carcinoma complicating liver cirrhosis.

Chemotherapy has been proposed for patients with hepatocellular carcinoma (HCC) associated with well-compensated cirrhosis who are unsuitable for locoregional treatments. Anthracyclines are the most active agents against HCC, although their toxicity is often unpredictable; thus, there is a need for new active drugs with a safe toxicity profile. The liposomal formulation of the anthracycline daunorubicin has low systemic toxicity and is taken up strongly by the liver. We started a phase I study with liposomal daunorubicin (starting dose 80 mg/m2 every 21 days) in patients with hepatocellular carcinoma and Child-Pugh stage A or B liver cirrhosis. At the starting dose, we recorded dose-limiting toxicities (1 hyperbilirubinemia, 1 prolonged prothrombin time, 1 persisting grade 3 neutropenia) in 3 out of 4 patients. Thus, according to protocol, we went down to the dose level of 60 mg/m2 but still 2 out of 3 patients had unacceptable toxicity (1 hypertransaminasemia, 1 hyperbilirubinemia with encephalopathia). Finally, we treated 4 more patients at the dose level of 40 mg/m2 and again we recorded liver toxicity in three of them. Overall haematological toxicity was mild and significant non-haematologic toxicities, other than hepatic, were not recorded. The toxicity profile observed warns against further assessment of liposomal daunorubicin in patients with hepatocellular carcinoma and liver cirrhosis.