European Society of Surgical Oncology's strategy for clinical research: Paving the way for a culture of research in cancer surgery.

As part of its mission to promote the best surgical care for cancer patients, the European Society of Surgical Oncology (ESSO) has been developing multiple programmes for clinical research along with its educational portfolio. This position paper describes the different research activities of the Society over the past decade and an action plan for the upcoming five years to lead innovative and high quality surgical oncology research. ESSO proposes to consider pragmatic research methodologies as a complement to randomised clinical trials (RCT), advocates for increased funding and operational support in conducting research and aims to enable young surgeons to be active in research and establish partnerships for translational research activities.

Metastasectomy in cutaneous melanoma.

Metastasectomy remains the only treatment in malignant melanoma to offer complete pathologic response within a few days of in-hospital stay. It has been historically associated with the highest survival rates in the literature reported for patients of this stage. However, only a minority of patients are amenable to curative resection of distant metastatic disease. This patient group exhibit slow growing oligometastases as indicated by: a. Long disease free interval after treatment of their primary tumours and b. An exhaustive preoperative work up with the use of PET/CT and MRI scans. Only complete resection of all metastases is associated with long term survival and debulking should not be attempted. Metastasectomy has also been shown to offer significant palliation in cases of gastrointestinal bleeding or obstruction. The timing and the sequencing of surgery in the modern multimodal targeted treatment of melanoma is still unclear and warrants further investigation.

Impact of varicocele on biological markers of gonadal function.

PURPOSE: The aim of this study is to evaluate the potential effect of varicocele in the hormonal and clinical profile of adolescents. METHODS: Twenty adolescents at Tanner stage 4-5 with left varicocele were studied and compared with a control group of 20 healthy adolescents. All patients underwent ultrasonographic testicular volumetry as well as hormonal evaluation of inhibin B, testosterone, baseline and gonadotropin-releasing hormone stimulated, follicle-stimulating hormone as well as luteinizing hormone. Statistical analysis was performed using the student's t test with p value <0.05 taken as statistical significant. RESULTS: Patients with varicocele showed reduced levels of inhibin B compared to controls and a significant reduction in the testicular volume on the affected side. The response of luteinizing hormone to gonadotropin-releasing hormone stimulation was significantly higher in the varicocele group compared to the control group. Furthermore a significant inverse relationship of inhibin B compared to follicle-stimulating hormone was noted. CONCLUSION: Serum inhibin B levels could represent a useful marker of Sertoli cell damage caused by varicocele.

Apoptosis and cell proliferation correlated with tumor grade in patients with lung adenocarcinoma.

BACKGROUND: Apoptosis and cell proliferation in patients with adenocarcinoma of the lung have not been well described with relation to fine-needle aspiration biopsies (FNABs). To investigate the contribution of apoptosis to the growth of adenocarcinoma of the lung, both apoptosis and cell proliferation were analysed for correlation with the grade of the tumor. PATIENTS AND METHODS: Fifty tumors from 50 patients with adenocarcinoma of the lung were studied. Twelve tumors were well-differentiated, 22 were moderately differentiated and 16 were poorly differentiated. The detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay was applied to investigate active cell death (apoptosis) and the MIB-1 antigen was used to investigate cell proliferation. RESULTS: The TUNEL indices were 0.55±0.09, 0.90±0.33 and 3.1±0.99 in well-, moderately and poorly differentiated adenocarcinoma of the lung respectively. The MIB-1 antigen labeling indices were 7.1±0.12, 14.3±3.5 and 28.7±6.9, respectively, in the same order of tumor differentiation. The differences in both TUNEL and MIB-1 labeling indices were significant between well-, moderately and poorly differentiated adenocarcinoma of the lung and a positive correlation was found between the TUNEL indices and the MIB-1 indices. CONCLUSION: Apoptosis (cell death) and cell proliferation increases as the grade of differentiation decreases in adenocarcinoma of the lung, suggesting a rapid turn over of the tumor cells in tumors with a lower grade of differentiation.

Image analysis of breast cancer immunohistochemistry-stained sections using ImageJ: an RGB-based model.

BACKGROUND: Image analysis of tissue sections using RGB image profiling is a modern accepted technique. MATERIALS AND METHODS: A new method of RGB analysis, using the freeware ImageJ, is presented which can be applied to sections with either nuclear or cytoplasmic staining. The step-by-step process is presented and the method is tested using breast cancer specimens immunostained for CK-19 and estrogen receptors. RESULTS: This image analysis easily discriminates CK-19 and estrogen receptor positivity in prepared breast cancer specimens. The method is easy to perform, without the need for previous image transformations. CONCLUSION: Compared to previous methods, this method proved more accurate in estimating the actual colours that an observer recognizes as positive after immunostaining. Further studies are needed to evaluate whether this method is efficient enough to be applied in clinical practice.

Breast cancer during pregnancy: a mini-review.

BACKGROUND: As modern women delay childbearing, pregnancy-associated breast cancer (PABC) becomes a more frequent problem faced by oncologists, gynecologists, and obstetricians alike. However, no evidence exists concerning the management of this condition. METHODS: We summarized the current literature regarding epidemiology, pathology, diagnosis, treatment and prognosis of PABC. Data were collected by searching PubMed and Medline for the period from 1950 to 2007. RESULTS: There are no randomized controlled trials regarding PABC management. Current evidence suggests that diagnosis may be carried out with limitations regarding staging; surgical treatment may be performed as for the non-pregnant women. Radiotherapy and endocrine therapy are contraindicated during pregnancy, while chemotherapy is allowed after the first trimester. Prognosis is considered poor. Subsequent pregnancy is allowed only 2 years after completing treatment. CONCLUSIONS: Due to lack of prospective randomized controlled clinical studies, both ongoing studies and future evidence are expected to solve problems related to breast cancer management during pregnancy.

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis.

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.

Correlation of heat shock protein (HSP70) expression with cell proliferation (MIB1), estrogen receptors (ER) and clinicopathological variables in invasive ductal breast carcinomas.

The aim of our study was to evaluate the relationship between the expression of HSP70 protein, cell proliferation, the expression of ER receptors and the clinicopathological variables Grade and LNS in breast invasive human tumors along with the role of HSP70 protein in the prognosis of human breast cancer. A strong association between HSP70 expression and ER content, in agreement with previous data, was found which revealed a statistically significant association between HSP70 positivity and ER expression (p<0.008) in 50 cases of invasive primary human breast cancers. We also found a strong correlation between HSP70 expression, Grade and LNS of invasive ductal breast carcinomas. This suggests that the expression of HSP70 plays a significant role in the progression of human breast cancer, and might prove useful in many other malignancies as an important marker for the outcome of the disease.

Expression of endothelial PDGF receptors alpha and beta in breast cancer: up-regulation of endothelial PDGF receptor beta.

The PDGF pathway is essential in tumor angiogenesis. Although the expression of the PDGF receptors has been excessively studied on breast cancer cells, few studies exist on PDGFR expression on the tumor endothelial cells. In the present study, it is investigated whether endothelial PDGF receptors' expression is altered in breast cancer. Endothelial PDGFRalpha and beta expression was initially studied under the influence of tumor conditioned medium derived from a breast cancer cell line. Following tissue culture experiments the endothelial expression of both receptors was studied on formalin-fixed paraffin-embedded tissue sections of normal breast and breast cancer specimens. The tissue culture experiment revealed a possible up-regulation of endothelial PDGFRbeta by breast cancer environment. Immunohistochemistry verified the result since 69.7% of the breast cancer sections were positive for PDGFRbeta compared to 43.3% of normal breast sections (p<0.05). No statistical difference was revealed by studying PDGFRalpha expression. In conclusion, our findings support the thesis of possible anti-PDGFRbeta anti-angiogenic therapy, in cases of endothelial PDGFRbeta-expressing breast cancer.

Highly conserved sequence of exon 15 BRAF gene and KRAS codon 12 mutation among Greek patients with colorectal cancer.

BACKGROUND: The RAS/RAF/MEK/MAP kinase pathway is essential to intracellular signaling transduction regulating cell proliferation, differentiation and death. We investigated the occurrence of exon 15 BRAF and KRAS codon 12 mutations among Greek patients with colorectal cancer. METHODS: Sixty-one samples from patients with sporadic colorectal adenocarcinomas were studied for exon 15 BRAF mutations. DNA from surgically resected specimens was analyzed by a combination of polymerase chain reaction and direct sequencing. KRAS codon 12 mutational analysis was technically possible in 58 samples (58/61) by a combination of polymerase chain reaction and restriction fragment length polymorphism. RESULTS: No exon 15 BRAF mutations were detected in any of the colon cancer specimens. The frequency of KRAS codon 12 mutations was 29.3% (17/58). Patients aged < or = 70 years more frequently presented carcinomas harboring KRAS codon 12 mutations than patients aged >70 years (p=0.028). Patients between 61 and 70 years of age were more likely to be carriers of this mutation (p=0.040). CONCLUSIONS: Despite the limited study sample, our data suggest that BRAF mutations might be present less frequently than KRAS mutations in Greek patients with colorectal carcinomas. Further research involving larger patient series will be necessary to confirm these findings and to assess possible ethnic, environmental and lifestyle influences on BRAF and KRAS mutagenesis.

Abdominal and pelvic stop-flow chemotherapy. Effect of chemotherapeutic agents and tissue ischemia on rectoanal pressures.

In hypoxic stop-flow chemoperfusion high doses of chemotherapeutic agents are almost directly administered to locally advanced tumors without risking significant systemic toxicity, although chemotherapy-induced neurotoxicity is still a problem. The aim of the study was to assess rectoanal motility and sensation before, during and after abdominal and pelvic stop-flow chemotherapy using the methods of stationary and ambulatory manometry. Stationary rectoanal manometry was performed within 24 hrs before and repeated 48 hrs after stop-flow chemotherapy in 7 consecutive patients with a history of locally advanced or recurrent abdominal and pelvic tumors. Anal sphincter resting and squeeze pressures, rectal sensitivity, rectoanal inhibitory reflex and rectal volumes at which temporary and permanent urge to defecate were reported were examined. Rectal volume associated with leak of rectal contents and rectal compliance were also assessed. Intraoperatively, changes in rectal and anal resting pressures before, during and after occlusion of the vessels and after administration of chemotherapeutic agent were as well recorded, analyzed and interpreted using ambulatory manometry. Induction of anesthesia reduced distal and proximal anal resting pressures. Vascular occlusion further and dramatically decreased resting pressures at all levels, which were fully recovered after re-establishing local blood circulation and for the rest of the recording period. Intraoperative administration of chemotherapy did not further affect anal resting pressures during or after hypoxia. No significant changes in rectoanal motility and sensation were detected on the 48 hrs postoperative assessment as compared to the preoperative state. Tissue hypoxia induced by vascular occlusion during stop-flow chemotherapy procedure, seems to be the only factor leading to a dramatic drop of anal pressures. Anal pressures fully recover after reperfusion of the isolated area. Furthermore, anorectal motility and sensation are not affected by any direct or indirect toxic action of the chemotherapeutic agents.

HSP-70, C-myc and HLA-DR expression in patients with cutaneous malignant melanoma metastatic in lymph nodes.

HSP-70, C-myc and HLA-DR were examined in patients with cutaneous malignant melanoma metastatic to lymph nodes. Lymph-nodal fine-needle aspiration biopsies (FNABs) were analyzed and the results were correlated to other variables, such as the gender of the patients, Clark level and Breslow thickness of the primary tumor. Thirty cases of metastatic melanoma in lymph nodes from 30 patients with cutaneous malignant melanoma were studied. All patients (100%) had microscopic regional nodal metastasis and a recurrence of the lesion during the first two years. The HSP-70, C-myc and HLA-DR expressions were investigated immunocytologically, using the APAAP (alkaline phosphatase) method on the FNAB samples. The immunocytochemical expressions of HSP-70 protein, C-myc oncogene, and HLA-DR antigen were found in 18 cases (60%), in 14 cases (43.3%) and in 12 cases (40%), respectively. Clark levels were significantly associated with HSP-70 protein (< 0.01), C-myc oncogene expression (< 0.05) and HLA-DR antigen (< 0.01) expression. The HLA-DR antigen was also found to be related (< 0.05) to higher Breslow thickness (> 1.5 mm). The clinical course of malignant cutaneous melanoma is related to the expression of these indices, which seem to play a significant role in the metastasis and prognosis of this aggressive tumor. The immunocytochemical expression of HSP-70 in the malignant melanoma tumor could be of particular value in the identification of patients with poor prognosis.

Modulation of vascular endothelium by imatinib: a study on the EA.hy 926 endothelial cell line.

In this study the EA.hy 926 endothelial cell line--simulating endothelial cells--was treated with imatinib in order to define a possible anti-angiogenic role for imatinib. Dose and time response experiments were performed. Cell morphology was studied, while migration efficiency, intercellular permeability and VE-cadherin expression were assayed, both in the presence and in the absence of imatinib. Imatinib-induced EA.hy 926 cell apoptosis was also examined. Results showed that imatinib reduced the endothelial cell population, changed cell monolayer morphology and reduced cell-to-cell cohesiveness. Migration efficiency was significantly decreased while intercellular permeability was 2.76-fold increased in the presence of imatinib. Indirect immunofluorescence microscopy showed nearly complete down-regulation of VE cadherin in imatinib-treated cells. Furthermore, apoptotic activity was detected in imatinib-treated cells. Altogether our results support an antiangiogenic profile for imatinib that possibly contributes to its therapeutic potential.

Telomerase activity as a marker of invasive ductal breast carcinomas on FNABs and relationship to other prognostic variables.

To study the activity of telomerase and the relationship between telomerase and other prognostic variables in cases of invasive ductal breast carcinomas, fifty fine-needle aspiration biopsies (FNABs) obtained from the same number of female patients, diagnosed cytologically and confirmed histologically after surgery, were examined. The same cases were studied immunocytochemically using monoclonal antibodies to telomerase, estradiol receptors (ER) and HER-2 (CB11) and a standard alkaline phosphatase (APAAP) method. Telomerase activity was found in 72% of the carcinomas studied. An association was found between telomerase activity, ER receptors and HER-2 expression (p <0.005). A relationship between telomerase activity, histological grade and lymph node status (LNS) was found as well (p<0.005). The above results seem to be significant prognostic factors and should be taken into consideration in the follow-up of patients after appropriate treatment for breast cancer.

Topoisomerase II alpha expression in breast ductal invasive carcinomas and correlation with clinicopathological variables.

Topoisomerase II alpha (topo IIa) is an enzyme that in normal cells is expressed predominantly in the S/G2/M-phase of the cell cycle. In malignant cells, in vitro studies have indicated that the expression of topo II alpha is both higher and less dependent on the proliferation state in the cell. To study the expression of topo IIa and the relationship between that expression-and other variables in cases of breast ductal invasive carcinomas, 50 fine-needle aspiration biopsies were performed from the same number of female patients, diagnosed cytologically and confirmed histologically after surgery. The same cases were studied immunocytochemically using monoclonal antibodies to topo IIa and Her2/neu (CB11) by the alkaline phosphatase method (APAAP). Topo IIa was found in 32 cases (64%) of the carcinomas studied. An overexpression between topo IIa and Her2/neu was found (p < 0.005). A relationship between topo IIa expression, histological grade and lymph node status (LNs) was also found (p < 0.005). Increased topo IIa expression seems to be related to an aggressive form of breast cancer featuring Her2 amplification and lymph node metastasis.

Endothelial p21(WAF1/CiP1) cell cycle inhibitor is down-regulated in breast cancer.

BACKGROUND: Tumor angiogenesis is considered a multi-pathway process, while p21(WAF1/CiP1) is a CDK inhibitor involved in cell division and survivaL Herein the tumor environment effect on endothelial p21(WAF1/Cip1) expression is examined. MATERIALS AND METHODS: The EA.hy 926 endothelial cell line and tumor-conditioned medium (TCM) from the MDA-MB-468 breast cancer cell line were used. Endothelial cells grown alone and in TCM were immunostained for p21(WAF1/Cip1) and analyzed by RT-PCR Forty-four cases of breast cancer and forty-three cases of normal breast tissue were immunostained for p21(WAF1/Cip1). RESULTS: Endothelial p21(WAF1/Cip1) is transcriptionally down-regulated under the influence of TCM. Moreover, it seems that breast cancer tumor endothelium does not express p21(WAF1/Cip1). CONCLUSION: P21(WAF1/Cip1) plays a major role in angiogenesis, since tumor cells seem to down-regulate endothelial p21(WAF1/Cip1), compared to endothelial cells grown in serum-free medium. The verification of the tissue culture experiment results by immunohistochemistry on tissue sections indicates p21(WAF1/Cip1) as a target of modern molecular therapy.

Establishment of a new co-culture model using negatively-charged slides.

Intercellular interactions are studied using different co-culture systems. Tumor conditioned medium-based systems, filter inserts and direct contact co-culture systems are often used according to research needs. In this article we present a new co-culture technique, using negatively-charged slides (NCS) as the culture surface. The technique was developed on a human-derived endothelial cell line-breast cancer interaction model. Two variations of this model are presented: In the first variation co-culture is achieved by using one NCS and a standard tissue culture treated dish as growing surfaces for the two cell populations respectively, while in the second variation the two cell populations are grown on two NCS. No significant difference was found between cell culture on the NCS compared to regular culture plasticware and staining was not only successfully but also easily performed. The suggested co-culture model has the advantage of allowing real time studies regarding cellular interactions. Additionally the two cell populations can be independently studied. Morphology is maintained throughout the procedure allowing morphological observation. Moreover, low cost is a factor permitting the application of the suggested model even in low budget laboratories. The features of the co-culture model that we developed are presented in relation to the salient features of other models.

Effect of sodium valproate on esophageal motility in healthy subjects and patients with gastroesophageal reflux.

BACKGROUND: There is experimental evidence to show that upper gastrointestinal tract motility is influenced by a GABAergic mechanism. Sodium valproate acts as a GABA agonist, and has been proven to affect the human internal anal sphincter. The aim of this study was to evaluate any possible effect of sodium valproate on esophageal motility in healthy subjects and patients with gastroesophageal reflux disease (GERD). METHODS: Ten healthy volunteers (4 M, 6 F; age range: 20-61 years) and 12 patients (4 M, 8 F; age range: 25-70 years) with GERD were included in the study. Standard esophageal manometry and ambulatory 24-h esophageal pH monitoring were performed before and 5 days after oral administration of sodium valproate (400 mg four times per day). Main measurements included a) lower esophageal sphincter (LES) resting pressure and amplitude and duration of peristalsis at 5, 10 and 15 cm proximal to LES, and b) percentage of time with esophageal pH <4 and number of reflux episodes. RESULTS: Sodium valproate (i) significantly increased LES resting pressure in both groups (P<0.05), without affecting either the LES postdeglutition relaxation or any of the parameters of the esophageal peristaltic activity, (ii) significantly reduced the number of reflux episodes at the postprandial period in both healthy subjects (P=0.02) and reflux patients without hiatal hernia (P=0.04) and (iii) the time percentage with esophageal pH <4 at the postprandial period in reflux patients (P=0.01). CONCLUSIONS: Sodium valproate increases normal and reduced tonic activity of the human LES and reduces the number of reflux episodes in health and GERD. This action could be attributed to a central GABAergic mechanism.

First-line intra-arterial chemotherapy (IAC) with epirubicin and mitoxantrone in locally advanced breast cancer.

BACKGROUND: Approximately 20% of patients with breast cancer present with locally advanced disease without distant metastases. This phase II double-center trial aimed at investigating the activity of epirubicin (Farmorubicin)--mitoxantrone (Onkotrone/Novantrone) combination as first-line intra-arterial chemotherapy (IAC) in locally advanced breast cancer patients. PATIENTS AND METHODS: Thirty-six patients with locally advanced disease and no prior exposure to anthracyclines received the following regimen: epirubicin (Farmorubicin) 30 mg/mq and mitoxantrone (Onkotrone/Novantrone) 10 mg/mq by IAC short infusion on day 1, every 3 weeks for up to six cycles. Prior to IAC an arteriogram of subclavian, internal mammary and lateral thoracic arteries was obtained in all patients, followed by infusion of a blue dye solution into the arteries to determine the most appropriate vessel that supplies the tumor area. RESULTS: Objective responses, confirmed at least 4 weeks after the first documentation, were observed in 25 patients (70%; 95%CI, 62% to 80%): 3 CR, 22 PR. Although three of the patients showed complete tumor regression, operative removal or toilet mastectomy became feasible in 25 patients since tumor shrinkage ranged over 75%. A total of 25 mastectomies were carried out for 36 patients. Four patients had bulky tumors (> 13 cm tumor diameter), while 8 patients had ulcerated tumors, two of which presented with complete infiltration of normal breast tissue. The median time to progression and median overall survival were 11 and 27 months, respectively. The time to local response was 3 weeks and time to mastectomy was 9 weeks. Transient neurological disorders developed in six patients and skin chemical burns with painful inflammatory reactions were encountered in ten patients. No systemic toxicity was observed in terms of bone marrow depression and hair loss. No cardiotoxicity was observed. In all specimens necrosis was reported (complete 3 cases, partial 16 and minimal 6). CONCLUSION: A combination of epirubicin (Farmorubicin) and mitoxantrone (Onkotrone/Novantrone) as IAC appears to be a safe and well tolerated treatment for locally advanced breast cancer without clinical evidence of distant metastases. When combined with surgery it offers interesting results in terms of local control and allows a high rate of mastectomies in otherwise inoperable cases.