Evaluation of thrombopoiesis kinetics by measurement of reticulated platelets and CD34+ cell subsets in patients with solid tumors following high dose chemotherapy and autologous peripheral blood progenitor cell support.

The transplantation of mobilised peripheral progenitor cells has resulted in shortening of neutrophil and platelet engrafment times following high-dose chemotherapy. Since reticulated platelet percentage (PR%) has been established as a measure of bone marrow platelet production, we performed this type of analysis on the thrombopoietic compartment during transplant-related chemotherapy. DESIGN AND METHODS: Kinetics of thrombopoiesis of 19 patients with solid tumors undergoing a single or double autologous peripheral blood progenitor cell transplant was characterized by evaluating the level of RP. The correlation between CD34(+) cell subsets and the time of highest percentage of RP was also evaluated. RESULTS: The percentage of RP increases since day +8 after single transplant reaching the peak (3.4%) at day +10. In the group of patients receiving double transplant, the RP value of peak observed after second transplant is not significantly different from that one observed after the first transplant (3 vs 3.7%). In a subgroup of patients both the number of CD34(+) cells/Kg infused and the percentage of CD34(+) CD61(+) cell subsets correlate with the day of RP peak. INTERPRETATION AND CONCLUSIONS: These results suggest that RP measurement is an early indicator of engraftment. Additionally, the observation that RP percentage is high at the time of platelet transfusion in 13 out of 20 cases of transfusions (the 7 cases with low RP value being transfused during the period of obligate thrombocytopenia) suggests that the evaluation of this parameter, together with the platelet count, can be used to monitor the need for platelet transfusion

Cardiac toxicity of trastuzumab in metastatic breast cancer patients previously treated with high-dose chemotherapy: a retrospective study

HER-2 overexpression is associated to a poor prognosis in high-risk and metastatic breast cancer (MBC) patients treated with high-dose chemotherapy (HDC). HER-2 status is also a predictive factor and when trastuzumab is administered in combination with or sequentially to chemotherapy, a significant disease-free and/or overall survival improvement has been observed in HER-2+ early and MBC. Unfortunately, in both settings, trastuzumab is associated with an increased risk of cardiac dysfunction (CD). We have reviewed the clinical charts of HER-2-overexpressing MBC patients treated with trastuzumab after HDC. Age, baseline left ventricular ejection fraction (LVEF), radiation therapy on cardiac area, exposure to anthracycline, single or multiple transplant, high-dose agents, trastuzumab treatment duration were recorded as potential risk factors. In total, 53 patients have been included in the analysis. Median LVEF at baseline was 60.5%; at the end of trastuzumab (data available for 28 patients only), it was 55% (P=0.01). Five out of the 28 (17.9%) patients experienced CD. Two out of 53 (3.8%) patients developed a congestive heart failure. Age ⩾50 years and multiple transplant procedure were potential risk factors for CD. The overall incidence of CD observed in this population of HER-2+ MBC patients treated with trastuzumab after HDC is not superior to that reported with concomitant trastuzumab and anthracyclines. However, patients with age ⩾50 years or receiving multiple course of HDC should be considered at risk for CD

Prognostic role of EGFR gene copy number and KRAS mutation in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy

Epidermal growth factor receptor (EGFR), evaluated by immunohistochemistry, has been shown to have prognostic significance in patients with colorectal cancer. Gene copy number (GCN) of EGFR and KRAS status predict response and outcome in patients treated with anti-EGFR therapy, but their prognostic significance in colorectal cancer patients is still unclear.We have retrospectively reviewed the baseline EGFR GCN, KRAS status and clinical outcome of 146 locally advanced rectal cancer (LARC) patients treated with preoperative chemoradiotherapy. Pathological response evaluated by Dworak's tumour regression grade (TRG), disease-free survival (DFS) and overall survival (OS) were analysed.Tumour regression grade 4 and TRG3-4 were achieved in 14.4 and 30.8\% of the patients respectively. Twenty-nine (19.9\%) and 33 patients (19.2\%) had an EGFR/nuclei ratio >2.9 and CEP7 polisomy >50\% respectively; 28 patients (19.2\%) had a KRAS mutation. Neither EGFR GCN nor KRAS status was statistically correlated to TRG. 5-year DFS and OS were 63.3 and 71.5\%, respectively, and no significant relation with EGFR GCN or KRAS status was found.Our data show that EGFR GCN and KRAS status are not prognostic factors in LARC treated with preoperative chemoradiation

Everolimus plus aromatase inhibitors as maintenance therapy after first-line chemotherapy: Final results of the phase III randomised MAIN-A (MAINtenance Afinitor) trial

Background: Despite endocrine therapy being the mainstay of treatment for hormone receptor positive (HR+)/HER2 12 metastatic breast cancer, patients at risk of visceral crisis or doubt for endocrine sensitivity are still offered first-line chemotherapy. Maintenance hormonal therapy is generally offered at the discontinuation of chemotherapy. The MAINtenance Afinitor study is a randomised, phase III trial comparing maintenance everolimus combined with aromatase inhibitors (AIs) versus AI monotherapy in patients with disease control after first-line chemotherapy. Methods: Patients with stable disease, partial response or complete response after first-line chemotherapy were randomised to everolimus plus AIs (exemestane or letrozole or anastrozole) or to AIs alone. Primary aim was progression-free survival (PFS). Secondary aims included response rate, safety and overall survival (OS). Results: In total, 110 patients were randomised to everolimus + AIs (n = 52) or to AIs (n = 58). Median PFS was 11.0 months (95% confidence interval [CI] 8.1\u201313.8) in the everolimus + AI arm and 7.2 months (95% CI 4.7\u201310.9) in the AI monotherapy arm (hazard ratio [HR] 0.71, 95% CI 0.47\u20131.06). Objective response rate was 22.4% in everolimus + AI arm and 19.2% in AI monotherapy arm. A higher proportion of disease progression as best response was reported in the AI monotherapy arm (28.8% versus 14.3%). Median OS was 35.7 months (95% CI 26.0\u201347.8) in the combination arm versus 33.5 (95% CI 26.4\u201342.7) in the AI alone arm (HR 1.0, 95% CI 0.61\u20131.62). Conclusions: EVE + AIs did not significantly impact on the outcome of metastatic breast cancer patients deemed suitable for first-line chemotherapy. Also taking into account treatment tolerability, maintenance endocrine therapy remains the standard. Trial registration: EudraCT: 2013-004153-24

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases

High-Dose Chemotherapy and Autologous Hematopoietic Stem Cell Transplantation as Adjuvant Treatment in High-Risk Breast Cancer: Data from the European Group for Blood and Marrow Transplantation Registry.

The aim of this retrospective study was to assess toxicity and efficacy of adjuvant high-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (AHSCT) in 583 high-risk breast cancer (BC) patients (>3 positive nodes) who were transplanted between 1995 and 2005 in Europe. All patients received surgery before transplant, and 55 patients (9.5%) received neoadjuvant treatment before surgery. Median age was 47.1 years, 57.3% of patients were premenopausal at treatment, 56.5% had endocrine-responsive tumors, 19.5% had a human epidermal growth factor receptor 2 (HER2)-negative tumor, and 72.4% had ≥10 positive lymph nodes at surgery. Seventy-nine percent received a single HDC procedure. Overall transplant-related mortality was 1.9%, at .9% between 2001 and 2005, whereas secondary tumor-related mortality was .9%. With a median follow-up of 120 months, overall survival and disease-free survival rates at 5 and 10 years in the whole population were 75% and 64% and 58% and 44%, respectively. Subgroup analysis demonstrated that rates of overall survival were significantly better in patients with endocrine-responsive tumors, <10 positive lymph nodes, and smaller tumor size. HER2 status did not affect survival probability. Adjuvant HDC with AHSCT has a low mortality rate and provides impressive long-term survival rates in patients with high-risk BC. Our results suggest that this treatment modality should be considered in selected high-risk BC patients and further investigated in clinical trials

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>mutations may predict poor response to radiotherapy. Downstream events from <it>KRAS</it>, such as activation of <it>BRAF</it>, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of <it>KRAS </it>and <it>BRAF </it>mutation status with p-AKT and p-ERK and outcomes in RC.</p> <p>Methods</p> <p>Pre-radiotherapy RC tumor biopsies were evaluated. <it>KRAS </it>and <it>BRAF </it>mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry.</p> <p>Results</p> <p>Of 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were <it>KRAS </it>WT, 95% were <it>BRAF </it>WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. <it>KRAS </it>WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by <it>KRAS </it>status.</p> <p>Conclusions</p> <p><it>KRAS </it>mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.</p

Sorafenib in patients with advanced biliary tract carcinoma: a phase II trial

BACKGROUND: Advanced biliary tract carcinoma has a very poor prognosis, with chemotherapy being the mainstay of treatment. Sorafenib, a multikinase inhibitor of VEGFR-2/-3, PDGFR-beta, B-Raf, and C-Raf, has shown to be active in preclinical models of cholangiocarcinoma. METHODS: We conducted a phase II trial of single-agent sorafenib in patients with advanced biliary tract carcinoma. Sorafenib was administered at a dose of 400 mg twice a day. The primary end point was the disease control rate at 12 weeks. RESULTS: A total of 46 patients were treated. In all, 26 (56%) had received chemotherapy earlier, and 36 patients completed at least 45 days of treatment. In intention-to-treat analysis, the objective response was 2% and the disease control rate at 12 weeks was 32.6%. Progression-free survival (PFS) was 2.3 months (range: 0-12 months), and the median overall survival was 4.4 months (range: 0-22 months). Performance status was significantly related to PFS: median PFS values for ECOG 0 and 1 were 5.7 and 2.1 months, respectively (P=0.0002). The most common toxicities were skin rash (35%) and fatigue (33%), requiring a dose reduction in 22% of patients. CONCLUSIONS: Sorafenib as a single agent has a low activity in cholangiocarcinoma. Patients having a good performance status have a better PFS. The toxicity profile is manageable

Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy

BACKGROUND: Cystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations. METHODS: We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity. RESULTS: We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC). CONCLUSION: Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%)