Veronique Cocquyt

The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

HER-2/neu as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel

AbstractPurpose. To evaluate the predictive value of HER-2 in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin (A) or with single-agent docetaxel (T). Experimental design. Patients from this study participated in a phase III clinical trial in which doxorubicin or docetaxel was administered for advanced disease. HER-2 was evaluated by IHC. In all positive cases, FISH was used to confirm the HER-2 positive status. The different cohorts of patients identified by HER-2 were examined to assess a possible relationship between HER-2 status and treatment effect. Results. Tumor samples were available for 176 of the 326 patients entered in the clinical trial (54%). HER-2 positivity was observed in 20% of the study population. A statistically significant interaction was found between response rates to the study drugs and HER-2 status, with HER-2 positive patients deriving the highest benefit from the use of T (odds ratio for HER-2 positive patients treated with T = 3.12 (95% CI 1.11–8.80), p= 0.03). The interaction between HER-2 and response rates to A and T was also confirmed by a multivariate analysis. No statistically significant interaction was found between HER-2 and drugs efficacy evaluated in terms of time to progression and overall survival, although in the HER-2 negative cohort A was at least as effective as T in term of overall survival. Conclusions. These results suggest that in HER-2 positive breast cancer patients docetaxel might be more active than doxorubicin, while in HER-2 negative patients doxorubicin might be at least as effective as docetaxel. Although the present results cannot have an impact on current practice, they allow us to formulate the hypothesis that HER-2 positive breast cancer is a heterogeneous disease with regard to sensitivity to anthracyclines and taxanes, and that this might be dependent upon other molecular markers including the p-53 and topoisomerase II alpha genes. This hypothesis is currently being tested prospectively in two different ‘bench to bed-side’ clinical trials.

Comparison of L-758,298, a prodrug for the selective neurokinin-1 antagonist, L-754,030, with ondansetron for the prevention of cisplatin-induced emesis

Substance P is localised in brainstem regions associated with emesis. Based on studies in the ferret, it was postulated that a neurokinin-1 (NK1) receptor antagonist would have antiemetic activity as monotherapy in humans receiving chemotherapy. L-758,298 is a water-soluble, intravenous (i.v.) prodrug for L-754,030, a potent and selective NK1 receptor antagonist. This double-blind, randomised, active-agent (ondansetron)-controlled study enrolled 53 cisplatin-naïve patients and evaluated the prevention of both acute (0-24 h) and delayed (days 2-7) emesis after cisplatin treatment (50-100 mg/m(2)). All patients received i.v. L-758,298 (60 or 100 mg) (n=30) or ondansetron (32 mg) (n=23) before cisplatin and efficacy was evaluated up to day 7 post-cisplatin. Nausea was assessed by means of a four-point ordinal scale at intervals over the 7 day period. In the acute period, the proportion of patients without emesis in the L-758,298 and ondansetron groups was 37 and 52%, respectively (no significant difference between the groups). Comparing the distribution of average nausea scores over the entire first 24 h revealed no significant difference between the groups. In the delayed period, the proportion of patients without emesis in the L-758,298 and ondansetron treatment groups was 72 and 30%, respectively (P=0.005). The distribution of average nausea scores in the delayed period was lower in the L-758,298 group compared with the ondansetron group (P=0.15 for the entire delayed period and P=0.043 for day 2 only). No serious adverse events were attributed to L-758,298. A single dose of L-758,298 substantially suppressed the delayed nausea and vomiting characteristic of high dose cisplatin and also appeared to reduce acute emesis post-cisplatin. The data also support the proposition that the underlying mechanism(s) of acute and delayed emesis are different.

Mutation analysis of P73 and TP53 in Merkel cell carcinoma.

The p73 gene has been mapped to 1p36.33, a region which is frequently deleted in a wide variety of neoplasms including tumours of neuroectodermal origin. The p73 protein shows structural and functional homology to p53. For these reasons, p73 was considered as a positional and functional candidate tumour suppressor gene. Thus far, mutation analysis has provided no evidence for involvement of p73 in oligodendrogliomas, lung carcinoma, oesophageal carcinoma, prostatic carcinoma and hepatocellular carcinoma. In neuroblastoma, two mutations have been observed in a series of 140 tumours. In view of the occurrence of 1p deletions in Merkel cell carcinoma (MCC) and the location of p73 we decided to search for mutations in the p73 gene in five MCC cell lines and ten MCC tumours to test potential tumour suppressor function for this gene in MCC. In view of the possible complementary functions of p73 and TP53 we also examined the status of the TP53 gene. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in four MCCs. In one MCC tumour, a mis-sense mutation located in the NH2-terminal transactivation region of the p73 gene was found. These results show that p73, analogous to neuroblastoma, is infrequently mutated in MCC. This is also the first report in which the role of TP53 in MCC has been investigated by sequencing the entire coding region of TP53. TP53 mis-sense mutations and one non-sense mutation were detected in three of 15 examined MCCs, suggesting that TP53 mutations may play a role in the pathogenesis or progression of a subset of MCCs. Moreover, typical UVB induced C to T mutations were found in one MCC cell line thus providing further evidence for sun-exposure in the aetiology of this rare skin cancer.

Better cosmetic results and comparable quality of life after skin-sparing mastectomy and immediate autologous breast reconstruction compared to breast conservative treatment

Preoperative chemotherapy (PCT) can be used in large primary breast cancer to facilitate breast conservative surgery (BCS). Cosmetic results of BCS are influenced by the size of the residual tumour, relative to the size of the breast. After mastectomy, immediate breast reconstruction (IBR) with autologous tissue provides excellent cosmetic outcome and has proven to be safe in breast cancer patients. Besides improving overall and disease free survival, Quality of Life (QoL), body image and cosmetic outcome are also important issues after treatment for breast cancer. In this study, Health-Related-Quality of Life (HRQL) and body image were evaluated, in patients treated with PCT, followed by BCS, or skin-sparing mastectomy (SSM) and perforator-flap breast reconstruction. Additionally, clinical observers assessed cosmetic outcome. All participants were evaluated by the Medical Outcomes Study (MOS) 36-item Short Form Health Status Survey (SF-36, 36 items) and a study-specific questionnaire. An external panel evaluated standardised photographs of the breasts. For all patients, norm-based scores of physical and mental health state are comparable with the general population, except for vitality (VT) score, which is somewhat lower. No significant differences can be observed between both groups. The majority of the patients were satisfied with the appearance of their breasts. The cosmetic results, assessed by the clinical team, were significantly better for patients having IBR, compared to BCS. The mean score was 7.5/10 for IBR, versus 6.0/10 for BCS (p<0.0001).Breast conserving treatment or mastectomy with reconstruction may yield comparable results of QoL, but cosmetic outcome is better after SSM and perforator-flap reconstruction. Patients must be offered both options, and clinicians should stress that both are equally effective.

Combined karyotyping, CGH and M-fish analysis allows detailed characterization of unidentified chromosomal rearrangements in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. Cytogenetic studies have indicated that deletions and unbalanced translocations involving chromosome 1 short arm material occur in 40% of the investigated cases. Recurrent chromosomal imbalances detected by comparative genomic hybridization (CGH) analysis were loss of 3p, 10q, 13q and 17p and gains of 1q, 3q, 5p and 8q. In order to study genomic aberrations occurring in MCC in further detail, we combined karyotyping, CGH and multiplex‐fluorescence in situ hybridization (M‐FISH), a strategy that proved to be successful in the analysis of other malignancies. Analysis of 6 MCC cell lines and 1 MCC tumor revealed mostly near‐diploid karyotypes with an average of 5 chromosomal rearrangements. The observed karyotypic changes were heterogeneous, with 3–27 breakpoints per case, leading to imbalance of the involved chromosomal regions that was confirmed by CGH. Chromosomal rearrangements involving the short arm of chromosome 1, the long arm of chromosome 3 and gain of 5p material were the most frequently observed abnormalities in our study. In keeping with previous observations, this series of MCCs showed no evidence for high‐level amplification. We provid a detailed description of chromosomal translocations occurring in MCC that could be useful to direct future intensive investigation of these chromosomal regions.

Cancer risk in immune-mediated inflammatory diseases (IMID)

Inflammation and cancer have a profound yet ambiguous relationship. Inflammation - especially chronic inflammation - has protumorigenic effects, but inflammatory cells also mediate an immune response against the tumor and immunosuppression is known to increase the risk for certain tumors.This article reviews current literature on the role of inflammation in cancer and the cancer risk in immune-mediated inflammatory diseases (IMIDs). We discuss the effect on cancer risk of different drug classes used in the treatment of IMIDs treatment, including biologicals such as tumor necrosis factor (TNF) inhibitors.Overall cancer incidence and mortality risk are similar to the general population in inflammatory bowel disease (IBD), and slightly increased for rheumatoid arthritis and psoriasis, with risk profiles differing for different tumor types. Increased risk for non-melanoma skin cancer is associated with thiopurine treatment in IBD, with the combination of anti-TNF and methotrexate in rheumatoid arthritis and with PUVA, cyclosporine and anti-TNF treatment in psoriasis. Data on the safety of using biologic or immunosuppressant therapy in IMID patients with a history of cancer are scarce.This review provides clinicians with a solid background to help them in making decisions about treatment of immune-mediated diseases in patients with a tumor history.This article is related to another review article in Molecular Cancer: http://www.molecular-cancer.com/content/12/1/86.

Use of Tamoxifen Before and During Pregnancy

For premenopausal patients with receptor-positive early breast cancer, administration of tamoxifen for 5 years constitutes the main adjuvant endocrine therapy. During pregnancy, tamoxifen and its metabolites interact with rapidly growing and developing embryonic or fetal tissues. Information about tamoxifen and pregnancy was gathered by searching PubMed. In addition, we had access to the records of the pharmaceutical company AstraZeneca. Because these observations are retrospective and other therapies and diagnostic measures are possible confounders, a causal relationship was not established between tamoxifen treatment and pregnancy outcome. The records from AstraZeneca documented three live births with congenital anomalies and four live births without congenital anomalies related to tamoxifen treatment before pregnancy. Tamoxifen therapy during pregnancy resulted in 16 live births with congenital malformations and a total of 122 live births without malformations. The 122 live births without malformations included 85 patients from a prevention trial that did not record a single anomaly, whereas the AstraZeneca Safety Database alone reported 11 babies with congenital malformations of 44 live births. Additionally, there were: 12 spontaneous abortions, 17 terminations of pregnancy without known fetal defects, six terminations of pregnancy with fetal defects, one stillbirth without fetal defects, two stillbirths with fetal defects, and 57 unknown outcomes. The relatively high frequency of severe congenital abnormalities indicates that reliable birth control during tamoxifen treatment is mandatory. After tamoxifen use, a washout period of 2 months is advisable based on the known half-life of tamoxifen. In case of an inadvertent pregnancy, risks and options should be discussed.

Pharmacokinetics of Intrvenous Alendronate

Alendronate is a potent bisphosphonate that has been studied for the treatment of osteoporosis and Pagets disease of the bone. To examine the pharmacokinetics of this drug, several groups of postmenopausal women were dosed intravenously in several studies. Twelve patients with metastatic bone disease were administered an intravenous dose of 10 mg of 14C‐labeled alendronate (∼26 μCi), and plasma, feces, and urine samples were collected for 72 hours. Radioactivity was excreted almost exclusively in urine, and all of it was accounted for by alendronate. Overall recovery accounted for 47% of dose, with the remainder presumed to be retained in bone. Metabolism of alendronate was not observed. Renal clearance of alendronate was 71 mL/min. An additional 10 subjects were given repeated IV administrations of alendronate to demonstrate that previous exposure does not alter the pharmacokinetic behavior of the drug. Examination of the findings from these and other studies in which alendronate was administered intravenously revealed that disposition of single doses is linear in the range of 0.125 to 10 mg. With the possible exception of a somewhat greater skeletal retention of a systemically administered dose, the pharmacokinetics of IV alendronate were found to be similar to those of other bisphosphonates.

Lobular carcinoma in situ and invasive lobular cancer of the breast.

Purpose of review The incidence of lobular carcinoma in situ and invasive lobular carcinoma of the breast is increasing. Recent data suggest that lobular carcinoma in situ is an indolent precursor for breast cancer, rather than a pure risk factor. This could imply free surgical margins become important. The risk of contralateral carcinoma and of multifocality of invasive lobular carcinoma is higher than for invasive ductal carcinoma. Therefore, the need for mastectomy, or even for preventative contralateral mastectomy is questioned. Conventional mammography or ultrasonography cannot always give useful preoperative information about the extent of lobular cancers. The value of dynamic contrast-enhanced magnetic resonance imaging needs to be established for these patients. Recent findings The risk of invasive carcinoma after lobular carcinoma in situ is increased. Invasive carcinoma is usually located at the index point of lobular carcinoma in situ and is of lobular histology. Dynamic contrast-enhanced magnetic resonance imaging can be useful in the detection and preoperative staging of invasive lobular carcinoma. The risk of local recurrence is high in patients with invasive lobular carcinoma. Mastectomy and breast reconstruction could be an option in selected patients. The response to preoperative chemotherapy is worse for invasive lobular carcinoma compared with invasive ductal carcinoma, with a greater need for rescue mastectomy. Summary Lobular carcinoma in situ and invasive lobular carcinoma are different entities from ductal carcinoma in situ and invasive lobular carcinoma. Their biological profile should be studied further in order to make the fine tuning of treatment possible. Abbreviations ALH: atypical lobular hyperplasia; BCS: breast-conservative surgery; DCE-MRI: dynamic contrast-enhanced magnetic resonance imaging; DCIS: ductal carcinoma in situ; IBTR: ipsilateral breast tumour recurrence; IDC: invasive ductal carcinoma; ILC: invasive lobular carcinoma; LCIS: lobular carcinoma in situ; MRI: magnetic resonance imaging; PCT: preoperative chemotherapy; VEGF: vascular endothelial growth factor.