Massimiliano Cariati

Alpha-6 integrin is necessary for the tumourigenicity of a stem cell-like subpopulation within the MCF7 breast cancer cell line

The identification of mammary epithelial stem cells raises the hypothesis that these cells may be crucial in the pathogenesis of breast cancer. To further support this, a highly tumourigenic sub‐population of cancer cells has recently been identified in primary and metastatic breast cancer samples. In this study, a sub‐population of cells displaying features normally attributed to stem cells was identified within the breast cancer cell line MCF‐7. This sub‐population is capable of growth in anchorage‐independent conditions as spherical organoids, displays resistance to proapoptotic agents and significantly greater tumourigenicity than its parental line, with as few as 1,000 cells able to form tumours in immunodeficient mice. Cells within this sub‐population can be enriched by serial passages in anchorage‐independence, and are characterized by over‐expression of the adhesion molecule α6‐integrin. Alpha‐6 integrin proves to be required for the growth and survival of these cells, as the knockdown of ITGA6 causes mammosphere‐derived cells to lose their ability to grow as mammospheres and abrogates their tumourigenicity in mice. These findings support the existence of a highly tumourigenic sub‐population in breast cancer cells. Furthermore, it shows α6‐integrin as a potential therapeutic target aimed at tumour‐generating subsets of breast cancer cells.

BEX2 Is Overexpressed in a Subset of Primary Breast Cancers and Mediates Nerve Growth Factor/Nuclear Factor-κB Inhibition of Apoptosis in Breast Cancer Cell Lines

We have identified a novel subtype of estrogen receptor (ER)-positive breast cancers with improved outcome after tamoxifen treatment and characterized by overexpression of the gene BEX2. BEX2 and its homologue BEX1 have highly correlated expression and are part of a cluster enriched for ER response and apoptosis genes. BEX2 expression is induced after estradiol (E2) treatment with a peak at 3 h, suggesting BEX2 is an estrogen-regulated gene. BEX2 belongs to a family of genes, including BEX1, NGFRAP1 (alias BEX3), BEXL1 (alias BEX4), and NGFRAP1L1 (alias BEX5). Both BEX1 and NGFRAP1 interact with p75NTR and modulate nerve growth factor (NGF) signaling through nuclear factor-kappaB (NF-kappaB) to regulate cell cycle, apoptosis, and differentiation in neural tissues. In breast cancer cells, NGF inhibits C2-induced apoptosis through binding of p75NTR and NF-kappaB activation. Here, we show that BEX2 expression is necessary and sufficient for the NGF-mediated inhibition (through NF-kappaB activation) of C2-induced apoptosis. We also show that BEX2 modulates apoptosis of breast cancer cells in response to E2 (50 nmol/L) and tamoxifen (5 and 10 micromol/L). Furthermore, BEX2 overexpression enhances the antiproliferative effect of tamoxifen at pharmacologic dose (1 micromol/L). These data suggest that a NGF/BEX2/NF-kappaB pathway is involved in regulating apoptosis in breast cancer cells and in modulating response to tamoxifen in primary tumors.

A novel model of dormancy for bone metastatic breast cancer cells

Mortality of patients with breast cancer is due overwhelmingly to metastatic spread of the disease. Although dissemination is an early event in breast cancer, extended periods of cancer cell dormancy can result in long latency of metastasis development. Deciphering the mechanisms underlying cancer cell dormancy and subsequent growth at the metastatic site would facilitate development of strategies to interfere with these processes. A challenge in this undertaking has been the lack of models for cancer cell dormancy. We have established novel experimental systems that model the bone microenvironment of the breast cancer metastatic niche. These systems are based on 3D cocultures of breast cancer cells with cell types predominant in bone marrow. We identified conditions in which cancer cells are dormant and conditions in which they proliferate. Dormant cancer cells were able to proliferate upon transfer into supportive microenvironment or upon manipulation of signaling pathways that control dormancy. These experimental systems will be instrumental for metastasis studies, particularly the study of cellular dormancy.

Intraoperative assessment of tumor resection margins in breast-conserving surgery using 18F-FDG cerenkov luminescence imaging: A first-in-human feasibility study

In early-stage breast cancer, the primary treatment option for most women is breast-conserving surgery (BCS). There is a clear need for more accurate techniques to assess resection margins intraoperatively, because on average 20% of patients require further surgery to achieve clear margins. Cerenkov luminescence imaging (CLI) combines optical and molecular imaging by detecting light emitted by 18F-FDG. Its high-resolution and small size imaging equipment make CLI a promising technology for intraoperative margin assessment. A first-in-human study was conducted to evaluate the feasibility of 18F-FDG CLI for intraoperative assessment of tumor margins in BCS. Methods: Twenty-two patients with invasive breast cancer received 18F-FDG (5 MBq/kg) 45–60 min before surgery. Sentinel lymph node biopsy was performed using an increased 99mTc-nanocolloid activity of 150 MBq to facilitate nodal detection against the γ-probe background signal (cross-talk) from 18F-FDG. The cross-talk and 99mTc dose required was evaluated in 2 lead-in studies. Immediately after excision, specimens were imaged intraoperatively in an investigational CLI system. The first 10 patients were used to optimize the imaging protocol; the remaining 12 patients were included in the analysis dataset. Cerenkov luminescence images from incised BCS specimens were analyzed postoperatively by 2 surgeons blinded to the histopathology results, and mean radiance and margin distance were measured. The agreement between margin distance on CLI and histopathology was assessed. Radiation doses to staff were measured. Results: Ten of the 12 patients had an elevated tumor radiance on CLI. Mean radiance and tumor-to-background ratio were 560 ± 160 photons/s/cm2/sr and 2.41 ± 0.54, respectively. All 15 assessable margins were clear on CLI and histopathology. The agreement in margin distance and interrater agreement was good (κ = 0.81 and 0.912, respectively). Sentinel lymph nodes were successfully detected in all patients. The radiation dose to staff was low; surgeons received a mean dose of 34 ± 15 μSv per procedure. Conclusion: Intraoperative 18F-FDG CLI is a promising, low-risk technique for intraoperative assessment of tumor margins in BCS. A randomized controlled trial will evaluate the impact of this technique on reexcision rates.

Age at diagnosis and distant metastasis in breast cancer--a surprising inverse relationship.

INTRODUCTION Predictors for site of distant metastasis and impact on survival in breast cancer are incompletely understood. METHODS Clinico-pathological risk factors for site of distant metastasis and survival were analysed in patients with invasive breast cancer treated between 1986 and 2006. RESULTS Of 3553 patients, with median follow-up 6.32years, 825 (23%) developed distant metastasis. The site of metastasis was bone in 196/825 (24%), viscera in 540/825 (65%) and unknown in 89 (11%). Larger primary invasive tumour size, higher tumour grade and axillary nodal positivity increased risk of metastasis to all sites. Lobular carcinoma was more likely to first metastasise to bone compared to invasive ductal carcinoma (NST). Oestrogen receptor (ER) negative, progesterone receptor (PgR) negative and/or Human epidermal growth factor (HER2) positive tumours were more likely to metastasise to viscera. A striking relationship between increasing age at diagnosis and a reduction in risk of distant metastasis to bone and viscera was observed. Median time to death from onset of metastatic disease was 1.52 (Interquartile range (IQR) 0.7-2.9)years for patients with bone metastasis and 0.7 (IQR 0.2-1.5)years for visceral metastasis. On multivariate analysis, despite the decrease in risk of distant metastasis with increasing age, there was an elevated hazard for death in patients >50years at diagnosis of metastasis if they developed bone metastasis, with a similar trend observed in the >70years age group if they developed visceral metastasis. CONCLUSION These findings indicate that there are biological mechanisms underlying the impact of age on the development of distant metastasis and subsequent death. This may have important implications in the treatment of breast cancer.

Adjuvant taxanes and the development of breast cancer-related arm lymphoedema

Despite affecting approximately one‐quarter of all patients undergoing axillary lymph node dissection, the pathophysiology of breast cancer‐related lymphoedema (BCRL) remains poorly understood. More extensive locoregional treatment and higher body mass index have long been identified as major risk factors. This study aimed to identify risk factors for BCRL with a specific focus on the potential impact of chemotherapy on the risk of BCRL.

Audit of local recurrence following breast conservation surgery with 5-mm target margin and hypofractionated 40-Gray breast radiotherapy for invasive breast cancer

INTRODUCTION The risk of ipsilateral breast tumour recurrence (IBTR) following breast conservation surgery (BCS) for invasive breast cancer (IBC) and radiotherapy is dependent on patient-, tumour- and treatment-related variables. In the Cambridge Breast Unit, breast conserving surgery has been performed with a target radial margin of 5 mm for IBC, in combination with 40-Gy hypofractionated (15 fractions) breast radiotherapy, since 1999. PATIENTS AND METHODS An audit was performed of cases treated between 1999 and 2004. A total of 563 patients underwent BCS for invasive breast cancer with 90.4% receiving radiotherapy (RT) and 60.4% of patients receiving boost RT (3 fractions of 3-Gy). RESULTS After a median follow-up of 58 months, five of the 563 (0.9%) patients developed IBTR. The 5-year actuarial IBTR rate was 1.1%. In terms of distant disease recurrence (DDR), 29 of the 563 (5.2%) had DDR during follow-up, giving a 5-year actuarial DDR rate of 5.4%. The 5-year breast cancer specific survival was 95%, with the poorer NPI groups having worse breast cancer specific survival (Log-rank, P<0.0001). More importantly, patients with IBTR had a shorter breast cancer-specific survival than those who were IBTR-free (Log-rank, P<0.0001). CONCLUSIONS Our treatment regimen, combining BCS with a 5-mm target margin and hypofractionated 40-Gy RT, results in an extremely low rate of IBTR, and compares favourably with the target IBTR rate of <5% defined by the Association of Breast Surgeons (ABS) at BASO guidelines.

Xenotransplantation of breast cancers.

Three experimental systems based on mouse models are currently used to study breast cancer: transgenic mice, carcinogen-induced models, and xenografts of breast cancers. Each of these models has advantages and limitations. This chapter focuses on xenotransplantation of breast cancers and reviews the techniques used so far in establishing this model, the advantages and limitations compared to other experimental systems, and finally, the technical questions that remain to be answered.

Internal mammary nodes and breast cancer

The lymphatic drainage pathway of the breast to the internal mammary nodes (IMNs) in humans was first described in 1786 and revised in 1790 by Cruikshank1. This concept has been confirmed in numerous studies and recently revisited in the era of sentinel lymph node (SLN) biopsy. Urban et al.2 reviewed 1000 consecutive patients with breast cancer treated between 1940 and 1945 by radical mastectomy and adjuvant radiation therapy. They observed a higher incidence of parasternal chest wall recurrences in those with medial tumours than in those with lateral tumours (10 versus 2 per cent). The authors thought these recurrences represented spread of metastatic disease to the IMNs and, although this was not a universally held view, they recommended extended radical mastectomy with dissection of IMNs as treatment for primary operable breast cancer2. The overall frequency of IMN metastasis in breast cancer is 22·5 per cent3, correlating strongly with the status of axillary lymph nodes (ALNs); IMNs are involved in 36 per cent of ALN positive patients, and in 9 per cent of ALN negative patients3. It may be argued that the high incidence of IMN involvement in ALN positive patients described in past years is now an overestimate; data from series of patients with relatively advanced disease should not be extrapolated to patients diagnosed and treated for early breast cancer. Still, recent evidence has shown that even those with early breast cancer have an incidence of IMN positivity of 17–27 per cent4–6. The relationship between the IMN status and site of tumour in the breast is controversial. Although Veronesi et al.7 described no relationship between tumour site and IMN positivity in a large series of patients, more recent evidence suggests such a link. Although the overall lymphatic drainage in breast cancer is predominantly to ALNs, some is to IMNs and medial tumours drain to IMNs more frequently than lateral tumours3. Factors increasing IMN positivity include a patient age less than 40 years, size of the primary tumour (16·0 per cent for tumours less than 2 cm and 24·5 per cent for larger tumours), and presence of concomitant ALN metastasis7. Veronesi et al.7 randomized 737 patients to Halsted mastectomy or extended radical mastectomy which included dissection of the IMNs. Although this trial showed no difference in overall and disease-free survival between the two groups, it nevertheless recognized the prognostic significance of the IMN status7. Ten-year overall and disease-free survival showed that patients fell into three groups based on nodal positivity. Those who were node negative in both ALNs and IMNs did better than those who were node positive in either ALNs or IMNs. Patients with IMN involvement alone had the same outcome as those with ALN involvement alone. However, patients with both nodal basins involved had the worst prognosis7. A further study by Sugg et al.8 showed IMN metastasis conferred a worse prognosis irrespective of axillary node involvement. It seems likely, therefore, that IMN involvement has significant prognostic importance in sub-groups of patients. Recent trials have shown a survival benefit in patients undergoing radiotherapy to the chest wall after mastectomy. Some have argued that part of this benefit may be attributed to irradiation of the IMNs9,10, but this remains unproven. An ongoing European randomized controlled trial (EORTC 22 922) is currently evaluating the impact of IMN and medial supraclavicular irradiation on longterm disease-free and overall survival in breast cancer patients with centrally or medially located tumours. Given the prognostic significance of nodal positivity in the internal mammary chain, it is reasonable to re-consider the surgical approach to these nodes. SLN biopsy allows accurate staging of the axilla and it is a technique that can be applied similarly to the IMNs. The internal mammary SLN can be detected only by techniques using radioisotope lymphoscintigraphy. The Netherlands Cancer Institute has shown recently in a series of 691 patients that the internal mammary SLN could be visualized on lymphoscintigraphy in 150 of them. Metastasis was detected in 22 (17 per cent) of these 150 patients, in whom the node was harvested. In nine of the 22, the IMN was the only positive node, the ALNs being negative. These 22 patients typify those who may benefit from adjuvant local radiotherapy, with or without systemic therapy5. Patients undergoing SLN biopsy as part of staging for early breast cancer are the group in which to examine the status of the IMNs. Those in whom

Diagnosis of right-sided varicocele: A retrospective comparative study between clinical examination, Doppler findings, US imaging and vascular anatomy at phlebography

Historically varicocele is diagnosed almost exclusively on the left side. The introduction of new imaging techniques has allowed the identification and characterization of right varicocele. This study aims to compare the diagnostic accuracy of various imaging techniques to data obtained using phlebography in the diagnosis of right varicocele. Patients treated for isolated right varicocele between 1992 and 2010 were retrospectively identified. Data from clinical examination, Doppler-USS, Color-Doppler-USS and Retrograde Phlebography were collected for each patient. 133 out of 4305 patients (3.1%) presented with an isolated right varicocele. 34 of these patients (25.6%) presented with palpable right varicocele. Doppler-USS identified various degrees of type I right venous reflux in 90 patients (67.7%). Phlebography showed venous reflux in all the patients (133), although with variability in terms of internal spermatic vein anatomy. Right varicocele is characterized by predictable anatomic features. Identification and characterization of these features is useful in guiding percutaneous treatment, allowing to optimize radiological display and reducing failure rate.