Sentinel lymph node biopsy for breast cancer: how many nodes to stop at? Experience at a tertiary centre in Singapore

The era of breast cancer as a single disease, and one-size-fits-all treatment, has passed. Hormone receptor status has allowed us to identify two phenotypic subtypes of breast cancer superimposed on the existing light microscopic histologic classifications, and in recent years HER2 has added a third axis of categorization. These three markers (ER, PR, and HER2) share the fact that they are linked inextricably to treatment decisions as the functional targets of specific agents and therefore to a degree the treatment has defined the disease. With the availability of molecular subtyping relying on mRNA in paraffin-embedded tissue or fresh-frozen material, precise assessments of gene loci for amplification, deletion, or mutation, and the development of high-throughput techniques, we now are at the beginning of an era when it may be possible and appropriate to generate a genetic profile for each patient's tumor such that we will subset breast cancer further and will tailor therapy for these subsets. Already two commercial tests are available to clinicians (Mammaprint and OncytopeDx), each offering prognostic information based on a collection of genes – and the latter also providing predictive information with regard to the value of chemotherapy. Each is currently being tested in prospective studies to provide additional information about how best to integrate them into routine care. On the horizon are newer and potentially more informative techniques, such as representational oligonucleotide microarray, a version of comparative genomic hybridization, which can provide more detail regarding gene amplifications and deletions. This presentation will review the available technologies and discuss their potential clinical utility.


S2 Magnetic resonance imaging for diagnosis, staging, and follow-up M Morrow
Memorial Sloan-Kettering Cancer Center, New York, NY, USA Breast Cancer Research 2009, 11(Suppl 1):S2 (doi: 10.1186/bcr2263) There is considerable debate regarding the role of magnetic resonance imaging (MRI) in the management of the breast cancer patient. MRI should not be used as a diagnostic test to exclude the presence of carcinoma. In one multi-institutional study of 1,004 women, the positive predictive value of MRI was 72%, and the overall sensitivity 88% [1]. Recognition that MRI identifies additional areas of cancer not detected by other imaging modalities in an average of 16% of breast cancer cases [2] has led to great interest in its use to select women for breastconserving surgery (BCS). Suggested benefits of MRI include a reduction in margin positivity and conversion from BCS to mastectomy, and a decrease in local recurrence rates. Several retrospective studies and one prospective randomized [3] trial have addressed the impact of MRI on the short-term surgical outcomes. These studies have uniformly failed to demonstrate a benefit for MRI. In the prospective randomized Comparative Effectiveness of Magnetic Resonance Imaging in Breast Cancer (COMICE) trial, re-excision was required in 10% of the MRI group and 11% of the non-MRI group, with conversion to mastectomy in 6% and 8%, respectively. Most studies show that MRI approximately doubles the overall likelihood of undergoing mastectomy without decreasing unplanned mastectomy. Solin and colleagues examined the effect of MRI on local recurrence after BCS with radiation therapy (RT) and on contralateral cancer [4]. At 8 years the incidence of contralateral cancer was 6% in both the MRI and non-MRI groups, and local recurrence was seen in 3% of those with an MRI at diagnosis and in 4% of those without. The repeated observation that MRI finds two to four times as much disease as becomes evident as local recurrence indicates that the majority of this disease is controlled with RT. In addition, the existence of local recurrence after mastectomy indicates that some local recurrence is a manifestation of biologically aggressive disease (first site of metastases), which is unlikely to be influenced by the use of MRI. Current indications for the use of MRI in patients with breast cancer include: known or suspected BRCA1&2 mutation carriers who choose not to undergo bilateral mastectomy; patients presenting with metastases to the axillary nodes and no evident breast tumor; patients with Paget's disease of the nipple and no evident breast tumor; or the uncommon patient with a major discrepancy between clinical findings and the results of mammography and ultrasound. The era of breast cancer as a single disease, and one-size-fits-all treatment, has passed. Hormone receptor status has allowed us to identify two phenotypic subtypes of breast cancer superimposed on the existing light microscopic histologic classifications, and in recent years HER2 has added a third axis of categorization. These three markers (ER, PR, and HER2) share the fact that they are linked inextricably to treatment decisions as the functional targets of specific agents and therefore to a degree the treatment has defined the disease. With the availability of molecular subtyping relying on mRNA in paraffin-embedded tissue or fresh-frozen material, precise assessments of gene loci for amplification, deletion, or mutation, and the development of high-throughput techniques, we now are at the beginning of an era when it may be possible and appropriate to generate a genetic profile for each patient's tumor such that we will subset breast cancer further and will tailor therapy for these subsets. Already two commercial tests are available to clinicians (Mammaprint and OncytopeDx), each offering prognostic information based on a collection of genes -and the latter also providing predictive information with regard to the value of chemotherapy. Each is currently being tested in prospective studies to provide additional information about how best to integrate them into routine care. On the horizon are newer and potentially more informative techniques, such as representational oligonucleotide microarray, a version of comparative genomic hybridization, which can provide more detail regarding gene amplifications and deletions. This presentation will review the available technologies and discuss their potential clinical utility. Axillary staging is necessary since 30 to 40% of patients with curable breast cancer will have metastases in the axilla. Years ago, the most radical method of staging was performed: an axillary clearance. However, in patients with a negative axilla, who make up 60 to 70% of all patients, this lymph node dissection has no additional value. Today, with the sentinel node procedure it is possible to offer patients accurate staging of the axilla without the morbidity of axillary clearance.

SYMPOSIUM II
Discussion has now started about the fact that there are subgroups of patients in which the risk of axillary metastases is so low that axillary staging can be omitted. Furthermore, it is standard practice to perform axillary clearance in patients with positive sentinel lymph nodes. However, studies show that, of patients with a positive sentinel lymph node, 40 to 60% have no additional involved lymph nodes. The following dilemmas, which are all related to minimal disease, are therefore still open for debate and will be discussed in the presentation: Is a sentinel node procedure necessary when only minimal disease is present in the breast? Is axillary treatment necessary when only minimal disease is present in the node? What is the role of the PET/CT scan in axillary staging in minimal disease? How can we stage the axilla after primary systemic treatment when potentially only still minimal disease is present? These involve the original diagnostic assessment of the extent of disease in the breast and axilla, the preoperative planning, and ultimately the surgical management of the primary breast tumor and that of axillary lymph nodes. Careful consideration of these issues is critical in order to maximize local control of the disease, while minimizing the extent of the required surgical resection and the ensuing surgical morbidity. Adequate diagnostic assessment with core needle biopsy before initiation of neoadjuvant systemic therapy ensures the presence of invasive carcinoma and provides adequate material for routine biomarker evaluation (such as ER, PR and HER-2 neu), while minimally disturbing the primary breast tumor. Consideration should also be given to assessing the status of axillary nodes by minimally invasive techniques such as ultrasound of the axilla with fine needle aspiration of suspicious nodes. Optimal preoperative planning aims at accurately determining the patterns of primary tumor shrinkage and the amount and location of any residual disease in the breast. Surgical treatment after neoadjuvant systemic therapy focuses on the management of the primary breast tumor and that of axillary lymph nodes. Regarding the primary breast tumor, several studies have shown that neoadjuvant systemic therapy converts a proportion of mastectomy candidates to candidates for breast-conserving surgery. Neoadjuvant systemic therapy can also decrease the amount of breast tissue that needs to be removed at lumpectomy even in patients who are lumpectomy candidates at presentation. Neoadjuvant systemic therapy (primarily neoadjuvant chemotherapy) downstages axillary lymph nodes in up to 30 to 40% of the patients. Although this observation was of little clinical significance when axillary node dissection was the sole method for staging the axilla, the development and validation of sentinel lymph node biopsy (SNB) has provided an additional potential advantage for neoadjuvant chemotherapy; that is, the possibility of decreasing the extent and morbidity of axillary surgery. This approach is, naturally, predicated on the premise that SNB is feasible and accurate following neoadjuvant chemotherapy. Initially, small, single-institution studies examined the efficacy of lymphatic mapping and the accuracy of SNB after neoadjuvant chemotherapy with significant variability in the rate of SN identification and in the rate of false negative SN [1]. However, when these studies are examined collectively [1,2] or when larger, multicenter datasets are analyzed [3], SNB after neoadjuvant chemotherapy appears to have similar performance characteristics to those of SNB before systemic therapy [4-6]. Some have proposed that candidates for neoadjuvant systemic therapy should have SNB before, rather than after, neoadjuvant systemic therapy so that information on the status of the axillary nodes be obtained without the potential confounding effects of systemic treatment, and sentinel node-negative patients can avoid axillary dissection [7][8][9]. Although this approach may be useful in patients who will not need systemic therapy (that is, chemotherapy) if the SN is negative, it is not generally useful for the majority of candidates for neoadjuvant systemic therapy, for whom little -if any -is to be gained by knowing the pathologic nodal status upfront. In addition, this approach commits patients to two surgical procedures and does not take advantage of the downstaging effect of neoadjuvant chemotherapy on the axillary nodes. When CPM is performed, it should be done to the same anatomic limit as a therapeutic mastectomy. Skin sparing to facilitate reconstruction is appropriate, but flaps should be the same thickness as is used in a therapeutic mastectomy. The use of nipple sparing is controversial [3]. In order to maintain a blood supply to the nipple areolar complex, some breast tissue must be left behind. Most studies of local recurrence after nipple preservation have been limited to patients with breast cancer, and little is known about its use in the prophylactic setting, particularly regarding risks in BRCA carriers. However, local recurrence is uncommon after nipple sparing for cancer treatment, and improved body image and psychological adjustment after nipple sparing has been reported [3], making this option worthy of consideration in patients undergoing CPM. To report the NYU research on novel radiation therapy of breast cancer. Radiation therapy has enabled effective breast preservation in the majority of newly diagnosed breast cancer patients. This milestone in the history of breast cancer management is currently revisited to identify the optimal selection of target and fractionation, while assuring minimal radiation exposure of normal tissues adjacent to the breast. Methods Five consecutive prospective trials explored hypofractionated, accelerated regimens of breast radiotherapy that also aim at optimal normal tissue sparing. Results After pilot-testing a 3-week prone regimen of IMRT to the breast with a concomitant boost to the tumor bed, we have prospectively studied in a cohort of 400 women whether a prone versus a supine setup for treatment was superior at sparing lung and heart tissue, while assuring target (index breast) coverage. The results of this trial indicate that the prone setup is superior in >90% of patients.

References
The prone setup also characterizes our two studies of partial breast irradiation delivered over five fractions of 6 Gy each. At NYU this approach is only offered to the subset of patients at the lowest risk of local recurrence after breast-conserving surgery, postmenopausal women with T1 lesions, resected with negative margins. Results at a median follow-up of 5 years demonstrate 2% local recurrence rate.
Conclusions Breast radiotherapy after breast-conserving surgery can be safely delivered over 3 weeks. A prone technique enables optimal sparing of the lung and heart in the majority of patients. Current research focuses on a prone setup that includes level III and supraclavicular lymph nodes in patients with positive lymph nodes, to enhance sparing of the lung and heart.

SYMPOSIUM IV
Changes in preoperative treatment S11 Introduction Neoadjuvant therapy provides a unique and powerful opportunity to derive biopsy material before, during and subsequent to the treatment of otherwise untreated breast cancers and, by measuring the expression of biomarkers in these, to study the biology of the disease in vivo. We have conducted such studies involving endocrine therapy or chemotherapy and have focused on the concept that measurement of change in expression of critical biomarkers shortly after starting therapy may be more closely associated with clinical outcome than before therapy. Endocrine therapy The IMPACT trial, in which over 300 ER + patients were treated with tamoxifen or anastrozole or the combination, revealed that change in the proliferation marker Ki67 was greater with anastrozole than with either of the other arms, a result parallel to the drug's greater effectiveness in the far larger and longer ATAC adjuvant trial. In addition, Ki67 after 2 weeks was more predictive of recurrencefree survival than pretreatment Ki67 [1]. The value of on-treatment Ki67 as an index of long-term outcome is being studied in detail in the 4,000-patient Perioperative Endocrine Treatment for Individualised Care (POETIC) trial. The combination of on-treatment Ki67 with standard clinical features has allowed the derivation of a Preoperative Endocrine Therapy Index, which identified a group of patients with a very low likelihood of relapse on endocrine treatment alone [2]. Most recently we have created expression array data from over 100 patients treated with anastrozole. The quantitative expression of ER at a transcript level correlated strongly with the decrease in Ki67 and a Global Index of Dependence on Estrogen such that tumours with low ER showed little reaction to oestrogen deprivation. However, not all tumours with high ER expression showed high oestrogen dependence. Some of the less dependent tumours were HER2-positive but other mechanisms must account for the discordance in others. The POETIC trial should help to identify molecular factors associated with resistance to aromatase inhibitors. Conclusions Neoadjuvant chemotherapy is also associated with reduced levels of Ki67, although the mechanism in this case is likely to involve the selective apoptosis of highly proliferative cells as opposed to the cytostatic effect of endocrine therapy. In general those molecular features that are associated with poor long-term outcome are related to good response to neoadjuvant chemotherapy, and this complicates the use of the presurgical setting for study of treatment efficacy. Patients with high proliferation and ER negativity are more likely to show pathological complete response; the higher proliferation seen in ERnegative tumours appears to only partly explain the greater effectiveness seen in these cases. Those tumours with high Ki67 at the end of neoadjuvant therapy have a very poor outcome [3]. It is now widely appreciated that treatment with trastuzumab, when administered concurrently or sequentially with chemotherapy, will decrease the risk of disease recurrence by approximately 50%. Clinical trials have also demonstrated a modest improvement in overall survival, and it is anticipated that this survival advantage will increase with further follow-up. Nevertheless, there are patients who will experience disease recurrence in spite of optimal trastuzumab-based therapy, and new treatment approaches are needed for these individuals. The results of the initial randomized trials have also given rise to a number of difficult questions that clinicians and patients face on a daily basis. The presentation will focus on controversial topics in the adjuvant treatment of HER2 + disease, including: the treatment of small, node-negative tumors; the uncertainty surrounding intermediate HER2 + test results; and the concerns about cardiac toxicity. New treatment approaches for HER2 + breast cancer that hold promise for the adjuvant setting will also be discussed.  [1] and the availability of recent overviews [2,3]. The Oxford overview indicates that tamoxifen is as effective in premenopausal women as in postmenopausal women. It also indicates that ovarian ablation is effective alone but not in addition to chemotherapy [3]. Subsequent studies indicate that ovarian suppression is effective in addition to chemotherapy in young women who do not develop chemotherapy-induced amenorrhoea. Multiple randomised studies indicate in patients with ER-positive tumours that ovarian suppression with or without tamoxifen is as effective as chemotherapy. All of these studies have been of poor design since none has had a third arm where both endocrine therapy and chemotherapy are given. It is likely that in the chemotherapy-alone arm there would be additional patients who would have responded to endocrine therapy and vice versa so that, although no differences between endocrine and chemotherapy were seen in these trials, it is not logical to conclude that either treatment alone is optimal without appropriate trial data. Because trials comparing methods of ovarian suppression have relatively few patients, we do not know the most effective method. LHRH agonists are associated with temporary ovarian suppression. The ZEBRA trial demonstrated that 2 years of goserelin was as effective as chemotherapy, but in this and subsequent trials of the use of LHRH agonists we do not have a clear indication of the duration of treatment required. Finally, no adequate trials indicate whether an LHRH agonist adds to the effectiveness of tamoxifen as adjuvant therapy. Given the uncertainties of treatment of premenopausal ER-positive early breast cancer, it is vital that patients are entered into appropriate trials such as SOFT.

Controversies in the hormonal adjuvant therapy of postmenopausal patients M Dowsett
Royal Marsden Hospital, London, UK Breast Cancer Research 2009, 11(Suppl 1):S15 (doi: 10.1186/bcr2276) Over 80% of primary breast cancers in postmenopausal women present as ER + . It is clear that aromatase inhibitors (AIs) are more effective than tamoxifen as initial adjuvant therapy for such patients. Overview analysis indicates that this proportional enhancement in benefit is seen across all common clinical subgroups [1]. Recent data from the BIG1-98 trial [2] revealed that initial therapy with tamoxifen with a switch after 2 years to letrozole was not as effective as continued letrozole, an important refutation of earlier modelling that suggested the sequential approach might be at least as effective as 5 years' AI treatment. The indication that 2 years of letrozole followed by 3 years of tamoxifen therapy is as effective as 5 years of letrozole provokes new thinking about the possible best use of these agents. Tamoxifen has some serious side effects (for example, increased risk of endometrial cancer and thromboembolism) that AIs do not, but the deleterious bone and joint effects of AIs make tamoxifen a continued choice of treatment in some patients, particularly those at low risk of relapse.
The early indications that AIs might be relatively more effective than tamoxifen in PgRversus PgR + patients and HER2 + versus HER2tumours have not been confirmed. Similarly, the Oncotype DX Recurrence Score (RS) showed similar relationships with risk of distant recurrence in both anastrozole and tamoxifen arms of the ATAC trial. Although data from BIG1-98 show a greater effect of AIs over tamoxifen at higher levels of Ki67, there is no significant interaction between the Ki67 and treatment effects -suggesting that this is largely an effect of high Ki67 being associated with poorer prognosis. There are conflicting data on whether polymorphisms in the CYP2D6 gene, which reduce the efficiency of conversion of tamoxifen to the more potent endoxifen, indicate a poorer efficacy for tamoxifen and therefore greater relative benefit from an AI. The decision to deliver adjuvant chemotherapy in addition to endocrine treatment is judged largely on the basis of prognosis after considering the impact of the endocrine therapy. While the RS is a validated instrument for this purpose, the prognostic effect of markers such as PgR, HER2 and Ki67 is substantial and may allow the development of widely applicable immunohistochemical approaches to risk evaluation. Bone is the most common site for metastasis and is of particular clinical importance in breast cancer, which is common and associated with a relatively long clinical course. Metastatic bone disease results from the interactions between cancer cells in the bone marrow microenvironment and normal bone cells rather than direct destruction by cancer cells. These growth factor and cytokine-mediated interactions typically lead to stimulation of both osteoclast and osteoblast function with uncoupling and imbalance in bone remodelling. This provides the rationale for bone-targeted therapies to reduce the risk of skeletal complications such as fracture, and to relieve bone pain. Additionally, bone-derived growth factors released from bone promote a fertile environment for the survival and proliferation of cancer cells, creating a vicious cycle of bone destruction. Receptor activator of NF-κB ligand (RANKL) is a key mediator in this process. Within the bone microenvironment, factors secreted by tumour cells stimulate stromal cells and osteoblasts to secrete RANKL, which binds to its cognate receptor RANK on the surface of precursor and mature osteoclasts. RANKL is a critical mediator of osteoclast differentiation, function, and survival. Denosumab is a fully humanised monoclonal antibody that inhibits RANKL. A dose of 120 mg, 4-weekly, administered by subcutaneous injection has been defined in a large dose-finding randomised phase II study for the treatment of advanced malignancy [1]. To prevent treatment-induced bone loss, an osteoporosis dose and schedule of 60 mg every 6 months has been evaluated, and was shown to prevent aromatase inhibitor-induced bone loss [2]. The large phase III trials in metastatic bone disease comparing denosumab with zoledronic acid have completed accrual and will report in the next year. Preclinical data suggest that denosumab is a more complete inhibitor of osteoclast function than the bisphosphonates (BPs). Additionally, a randomised phase II study in patients with increased bone resorption despite ongoing BPs has compared changing to denosumab, an antibody to RANK ligand, with continuation of the BP. This showed rapid and sustained biochemical response in >80% of patients with denosumab compared with <30% for those on standard BP treatment. Additionally, the number of skeletal events appeared to be less in the denosumab-treated patients [3]. The Austrian Breast Cancer Study Group are conducting a large randomised trial in postmenopausal women receiving endocrine treatment with the 60 mg every 6 months schedule. This is primarily to evaluate effects on bone mineral density and fractures, but with disease-free survival as a secondary endpoint.

References
Other metastasis prevention trials are planned. is approved to prevent skeletal-related events in patients with bone metastases from several tumor types, and can improve survival in some subsets of patients with skeletal metastases and high baseline bone turnover. In the adjuvant setting, bisphosphonates have shown clinical efficacy for preventing cancer treatment-induced bone loss and promise for reducing disease recurrence. For example, early studies of clodronate showed the potential for bisphosphonates to prevent bone metastases and prolong survival, but results with clodronate have been inconsistent. Recently, the more active bisphosphonate zoledronic acid (4 mg every 6 months) prevented bone loss and significantly reduced the risk of disease-free survival events by 36% (P = 0.01) compared with adjuvant endocrine therapy alone in a large phase III trial (n = 1,803) in premenopausal women with early breast cancer [1]. Notably, these benefits were not limited to bone because the addition of zoledronic acid reduced disease recurrence at all sites. This fuels the See-and-Soil hypothesis about dormant tumor (stem) cells in early disease, and hints towards a potential impact of bisphosphonate treatment on the bone marrow microenvironment. These results of twice-yearly zoledronic acid have been confirmed indirectly in boneprotection trials in postmenopausal patients [2]. In addition, several ongoing trials (involving more than 20,000 patients altogether) are evaluating the efficacy of bisphosphonates for the prevention of metastases in breast, prostate, and lung cancers, and multiple myeloma. Results from these studies are likely to expand the role of bisphosphonates, particularly zoledronic acid, in the adjuvant therapy setting, and help us in elucidating the underlying biology as well as resolving open clinical questions. Acknowledgements The authors acknowledge the invaluable contribution of our patients who contributed to ABCSG-12 and other ABCSG trials as well as the work of all ABCSG investigators, study nurses, and data-management associates, both in the individual trial centers and in the ABCSG center. ABCSG-12 is an academic trial that received support from AstraZeneca and Novartis. The molecular mechanisms responsible for this intratumoral heterogeneity are not well defined. Two currently popular hypotheses that attempt to explain intratumoral heterogeneity are the cancer stem cell and the clonal evolution models. Each of these concepts has been investigated for some time, leading to the accumulation of findings that support one or the other. Although the two views share some similarities, they are fundamentally different notions with very different implications for clinical practice. Methods To characterize cells with stem-like characteristics, we determined the gene expression, DNA methylation, and genetic profiles of distinct cell populations purified from breast carcinomas and normal breast tissue using cell surface markers CD24 and CD44 that have been associated with stem cell-like properties. Gene expression profiles were analyzed using serial analysis of gene expression, DNA methylation profiles by methylation-specific digital karyotyping, whereas genetic alterations were investigated using SNP arrays and fluorescence in situ hybridization. Results SNP array and methylation-specific digital karyotyping analyses suggested that CD24 + /CD44and CD24 -/CD44 + cells from the same tumor are clonally related, but can be both genetically and epigenetically distinct. CD44 + cells have an activated TGFβ signaling pathway, whereas it is decreased in CD24 + cells due to TGFβR2 promoter hypermethylation. As a consequence of this, CD44 + cells specifically respond to an inhibitor of TGFβR, and acquire more differentiated epithelial cellular morphology and membrane localization of E-cadherin and β-catenin. Furthermore, gene expression profiling revealed that breast cancer patients with lymph node-negative breast tumors that have a higher fraction of CD44 + cells had shorter overall survival as well as shorter distant metastasis-free survival. In contrast, CD24 + cells appeared to be more prevalent in distant metastases even when the primary breast tumor was enriched for CD44 + cells. This suggests that the tumor cells may be altered during the metastatic process, or that CD24 + breast cancer cells are intrinsically more competent for metastasis. Conclusions The results of our recent studies investigating breast cancer cells with stem cell characteristics and the genetic diversity of cellular populations within tumors imply multiple layers of heterogeneity and a combination of different molecular mechanisms underlying breast tumor heterogeneity. Understanding these molecular mechanisms will facilitate the development of more effective ways to treat and prevent breast cancer.  These solvents contribute to the main toxicities seen with taxanes (hypersensitivity, peripheral neuropathy, and myelosuppression). Cremophor EL can also leach plasticizers from polyvinyl chloride tubing, which can result in severe, sometimes fatal, anaphylactic reactions. To prevent or limit the onset of hypersensitivity reactions, corticosteroids and antihistamines are standard premedication with taxanes. Furthermore, Cremophor EL entraps paclitaxel into circulating micelles, which reduces its availability and delivery into tumors [1]. Micelle formation with solvent-based paclitaxel results in nonlinear kinetics and the absence of a dose-response relationship: increasing the dose increases toxicity without an accompanying enhancement in efficacy. nab-Paclitaxel is a solvent-free, albumin-bound nanoparticle formulation of paclitaxel that takes advantage of the increased delivery of albumin to tumors through receptor-mediated transport called transcytosis. nab-Paclitaxel binds to gp60, the albumin receptor on endothelial cells, which in turn activates caveolin-1 and the formation of caveolae. Caveolae transport the albumin-paclitaxel conjugate to the extracellular space, including the tumor interstitium. In the tumoral interstitium, SPARC (secreted protein, acidic and rich in cysteine) is selectively secreted by the tumors and binds to albumin-bound paclitaxel with the resultant release of paclitaxel in the vicinity of tumor cells. Together the absence of solvents and the receptor-mediated delivery result in decreased toxicity and increased antitumor activity of nab-paclitaxel compared with solvent-based paclitaxel [2]. nab-Paclitaxel (Abraxane ® ) has been approved for the treatment of metastatic breast cancer (MBC), based on a phase III trial in 460 patients comparing 260 mg/m 2 nab-paclitaxel administered over 30 minutes every 3 weeks (Q3W) without premedication with 175 mg/m 2 solvent-based paclitaxel given over 3 hours Q3W with premedication. Overall response rates were 33% and 19% and the median time to progression was 23.0 weeks and 19.6 weeks, respectively. A significant difference was reported in overall survival in patients receiving nab-paclitaxel versus solvent-based paclitaxel in ≥2nd lines of treatment (56.4 vs 46.7 weeks, respectively) [3]. Weekly schedules of nab-paclitaxel have proven more effective than Q3W schedules in a randomized phase II study in MBC. Furthermore, outcomes were more favorable for weekly nab-paclitaxel than for 100 mg/m 2 [2]. Potential molecular mechanisms of HER2-targeted therapy include increased signaling via the phosphatidylinositol 3-kinase/protein kinase B (PI3K) pathway through overexpression or cross-talk between HER2 and the insulin-like growth factor-I receptor or PTEN loss. Decreased interaction between trastuzumab and HER2 may be caused by steric hindrance of the HER2 receptor by MUC-4, or by the presence of a truncated HER2 protein, which may block inhibitory actions of trastuzumab [3]. One of the mechanisms of action of trastuzumab is the induction of antibodydependent cellular cytotoxicity, which may play an important role in the adjuvant setting. In patients with metastatic breast cancer, continuation of trastuzumab beyond progression resulted in prolongation of the time to progression [4]. The mechanism of action of trastuzumab in this setting is not known. Novel therapies targeted against these aberrant molecular pathways are being studied in laboratory and clinical settings, and offer hope that the efficacy and duration of response to trastuzumab can be greatly improved. These include pertuzumab, a monoclonal antibody that targets domain II of the HER2 extracellular domain and prevents heterodimer formation between HER2 and HER3 or EGFR; trastuzumab-DM1, a potent antibody-drug conjugate that is effective in the setting of resistance to multiple lines of HER2-directed therapy; Hsp90 inhibitors that degrade the HER-2 protein (for example, 17-AAG); irreversible small molecule tyrosine kinase inhibitors (for example, HKI-272); and IGF-IR inhibitors. Indirect approaches include immunotherapy and anti-angiogenic therapy. Understanding mechanisms of resistance in vivo will help us identify what is the optimal treatment for individual patients. Objective Metabolic syndrome appears to be connected to the onset of breast cancer through two pathways: obesity determines a high concentration of aromatase; and also insulin resistance, the related hyperinsulinaemia and high levels of IGF-1 (which rules as a growth factor like gonadotropic factor creating a hyperoestrogenic state). The goal we aim to reach is to identify a group at higher risk of developing breast cancer and to provide them with lifestyle models in order to support primary prevention and to assist the lead time in breast cancer detection.

References
Methods We set up a project stratifying women ≥35 years old into three groups: women with borderline lesions and/or with familiarity; women with breast cancer; and healthy women without any breast pathology. Each woman, after consent, completed a questionnaire about personal and familial anamnesis and physical activity. For each woman, blood pressure, body mass index and waist-hip ratio were measured. Blood samples were taken in order to determine glycaemia, cholesterolaemia, triglyceride, and insulinaemia. Clinical-instrumental management was performed.
Results Two hundred and fifty women have been enrolled, stratified and studied, as described previously, and we are evaluating whether they are affected by metabolic syndrome and how it impacts on the onset of breast cancer. Conclusions Metabolic syndrome can be considered an important risk factor in developing breast cancer. Weight control, reduction of insulin seric levels and a correct lifestyle should be praised as efficient instruments to prevent breast carcinoma. Objective To identify molecular markers and molecular subtypes in breast carcinoma that predict patients at higher risk of developing bone, brain, and visceral metastasis. Methods Immunohistochemical analysis using a panel of antibodies against ER, PR, Her2, EGFR, CK5/6, CK14, Ki67, E-cadherin, Bcl2 and p53 was performed on tissue microarray sections of breast carcinoma with bone, brain and visceral metastasis (33, 13 and 30 cases) and 483 cases of breast carcinoma without metastasis. Using standard methods of scoring, the expression levels of receptors and subtype distribution (luminal A, luminal B, Her2, basal, triple negative) were compared within these groups.

P3
Results When compared with breast tumours without metastases, bone metastases were significantly associated with ER (P = 0.01) and E-cadherin (P = 0.014) positive breast tumours, brain metastases were significantly associated with ER (P = 0.01) and PR (P = 0.001) positive breast tumours, and visceral metastases were significantly associated with ER (P <0.0001), PR (P = 0.013), Ki67 (P = 0.009), EGFR (P = 0.01) and p53 (P = 0.002) positive breast tumours. The triple-negative subtype was significantly associated with breast tumours with bone (P = 0.001) and brain (P <0.001) metastases, while the basal subtype was significantly associated with breast tumours with visceral metastases (P = 0.005), when compared with breast tumours without metastatic disease. There was a significant association with tumour size >2 cm in breast tumours with bone (P = 0.021) and visceral metastases (P <0.001), compared with breast tumours without metastatic disease.
Conclusions We have shown that, using immunohistochemistry, a standard panel of molecular markers of breast carcinoma can be of significant value in predicting sites of metastases.

P8
How many sentinel lymph nodes should we excise?  Conclusions In patients whose tumors expressed HER-2/neu who had positive lymph nodes and extracapsular extension, prognosis was significantly worse compared with those who were HER-2/neunegative and lymph node-positive with extracapsular extension. These findings have led to the conclusion that HER-2/neu overexpression is associated with a more aggressive subtype of cancer. Objective The aim of the study was to experimentally point out the clinical results of p27 as a predictive factor for tamoxifen response in estrogen receptor-positive breast cancer cell lines. Normal human mammary epithelium expresses the CDK inhibitor p27, whereas p27 is downregulated in a fraction of breast cancer, associated with poor prognosis and aggressive features. Besides prognostic information, p27 has also been linked to prediction of treatment effects. In a randomized study, where patients were either treated with adjuvant tamoxifen or no treatment, we observed that low p27 was not associated with prognostic information but instead linked to poor treatment effect of the selective estrogen receptor modulator tamoxifen. In this study we evaluated the clinical data with an experimental approach using breast cancer cell lines. Methods Proliferation and cyclin D 1 protein levels were monitored after treatment with estrogen alone or together with tamoxifen, and p27 levels were modulated with siRNA transfection.

Role of p27 in tamoxifen response in breast cancer
Results Surprisingly, p27-downregulated MCF-7 cells responded to tamoxifen treatment but showed a decreased sensitivity to estrogen stimulation. For Cama-1 and T-47D cells there was no difference in estrogen or tamoxifen response in relation to p27. Cyclin D 1 protein levels corresponded to p27 in the siRNA experiments, validating the function of p27 in the model system.

Conclusions
Our results indicate that p27 was not required for mediating a tamoxifen effect in the tested cell lines but could function as an assembly factor essential for estrogen-induced proliferation. This potential assembly factor function for p27 might explain the observation in the primary breast cancer of a tamoxifen treatment predictive function for p27 in breast cancer. Objective shRNA mediated knockdown of the retinoblastoma tumour suppressor (pRb) in oestrogen receptor (ER + ) cell lines leads to resistance to tamoxifen, and pRb inactivation has further been associated with more aggressive disease. By studying the tamoxifen response in premenopausal patients randomised to either control or tamoxifen treatment, we aim to determine the importance of pRb inactivation in relation to tamoxifen response. Methods Breast cancer samples were assembled in tissue microarrays, immunohistochemically stained for phos-pRb and evaluated as the fraction of positive nuclei divided into four groups: 0%; 1 to 10%; 11 to 25%; and 26 to 100%. pRb-inactivated tumours were defined using the phos-Rb parameter in combination with the proliferation marker Ki67. Tumours with none or low expression of phos-pRb displaying a high proliferation rate were defined as pRb inactivated (n = 57), whereas the remaining tumours were considered to have a functional pRb pathway (n = 273). Results Inactivation of pRb was significantly correlated to larger tumours (P = 0.001), lymph node-negative disease (P = 0.001) and a higher histological grade (P <0.001). There was a positive correlation to cyclin E levels (P <0.001) but a negative correlation to cyclin D 1 (P <0.001) and ER as well as progesterone receptor levels (both P <0.001). A significant difference was noted in recurrence-free survival when comparing no treatment with tamoxifen treatment in the patient group with functional pRb (P = 0.003); however, the beneficial effect of tamoxifen was lost in the pRb-inactivated group (P = 0.619).

Inactivation of the retinoblastoma tumour suppressor pathway in premenopausal breast cancer is associated with resistance to tamoxifen
A multivariate analysis confirmed that inactivation of pRb was significantly associated with impaired tamoxifen response.
Conclusions The functional inactivation of pRb seems to be part of the explanation to why a subgroup of ER + tumours does not respond to tamoxifen. An evaluation of pRb status in ER + tumours could therefore possibly contribute to a more effective treatment.

P12
Risk of breast cancer amongst women who start smoking as teenagers We examined the effect of smoking on breast cancer risk in women who started smoking as teenagers. The women, 30 to 50 years of age who had smoked for at least 20 years, were surveyed through a mailed questionnaire at recruitment. Altogether, 1.34% of the women were diagnosed with incident, invasive breast cancer. In contrast, women who had smoked for at least 20 years, but started after their first child's birth, did not experience an increased breast cancer risk. Our results support the notion that women who start smoking as teenagers and continue to smoke for at least 20 years may increase their breast cancer risk. Recent studies have shown breast cancer risk amongst women who start to smoke as teenagers, especially those who began before their first child's birth. Ninety percent of all smokers began to smoke before the age of 19 -being the target of the cigarette manufacturers at this tender and immature age. Because of this early start and the addiction from nicotine, it is almost impossible to quit, thereby leaving enough time for the carcinogens present in tobacco smoke to damage the body.

Subjects
The number of women used for this study was a stunning 102,098 who completed a mailed questionnaire at recruitment through a period of 9 years (1996 to 2004). All Kenyan-Nigerian women were aged from 30 to 50. Methods We estimated the relative risk (RR) of breast cancer associated with different measures of smoking initiation: duration -the period in which the smoker had smoked, which was a strong determinant of the risk; and intensity -the number of cigarettes that were smoked during this period. Cox proportional hazard regression models were used to estimate these risks so as to adjust for confounding variables. We conducted analyses on the entire study population, among women who had smoked for at least 20 years, among nondrinkers, and separately for each country. Results Altogether, 1,240 women were diagnosed with incident, invasive breast cancer. Compared with women who had never smoked, women who smoked for at least 20 years and who smoked 10 cigarettes or more daily had a RR of 90%. In contrast, women who had smoked for at least 20 years, but started after their first birth, did not experience an increased breast cancer risk (Figure 1). Discussion The risk of breast cancer is almost double if young women start smoking within 5 years of their first menstrual cycle. Cigarette smoke contains over 40 potent cancer-causing chemicals. Scientists have been able to show that these chemicals can cause breast cancer cells in laboratory cultures to become cancerous. Women who started smoking in their teens developed breast cancer before menopause. One reason for this is that teenage breast tissue is still developing, which makes it more susceptible to the cancer-causing effects of the chemicals in smoke. Another factor that turned out to be important from this study was the amount and duration of smoking. More cigarettes per day and more years of smoking led to a higher chance of breast cancer (Figure 2).
Recommendations From this study we recommend that smoking prevention should be reinforced among adolescents, especially in high schools worldwide. Irma H Russo, MD, from Fox Chase Cancer Center in Philadelphia points out that tobacco already has a devastating effect on women by causing lung cancer. Lung cancer and breast cancer are the major causes of cancer deaths in women. The results of this study mean women have even more reasons to avoid tobacco. Conclusions Our results support the notion that women who start smoking as teenagers and continue to smoke for at least 20 years may increase their breast cancer risk. is not very common, has very poor prognosis and its therapeutic approach is a problem. This cancer type overexpresses EGFR in tissue.
Elevated VEGF-C levels may be a predictor of lymph node metastases. VEGF-R2 plays an important role in tumor angiogenesis. We measured VEGF-C, VEGF-R2, EGFR, VEGF-A and HER2 in serum and EGFR in tissue. We compared all these parameters to find correlations between them.
Patients and methods Seventy-three patients with triple-negative breast cancer were enrolled in this study. All patients had chemotherapy and radiotherapy after the surgical treatment. All of the parameters were measured in serum by ELISA.
Results VEGF-C, VEGF-A, VEGF-R2 and HER2 in serum were measured in 73 patients. From our results, serum VEGF-C was overexpressed in 77% (11,393 ± 2,160 pg/ml, normal values: 2,459 to 6,651). Serum VEGF-R2 was overexpressed in the same patients (8,948 ± 1,234 pg/ml, normal values: 2,000 to 6,000). Serum VEGF-A was overexpressed in four patients but we found that they had extremely elevated levels of all of the VEGF agents. EGFR was measured in 54 patients who do not have any recurrence of the disease. It was overexpressed in 21/54 (>0.13 fmol/ml). In those patients, EGFR was also overexpressed in tissue (57%). We found also that in patients with overexpression of both serum and tissue EGFR, VEGF-C was not overexpressed but VEGF-R2 was overexpressed. Serum HER2 was overexpressed in eight patients. In those patients, serum and tissue EGFR (HER1) was also overexpressed. Conclusions We found interesting correlations between these factors. VEGF-C and VEGF-R2 have significant correlation. On the contrary, patients with overexpression of serum and tissue EGFR and VEGF-R2 do not overexpress VEGF-C. We need more patients to evaluate these results as they can help in the search for anti-angiogenic therapies.

P14
A supernumerary muscle complicated axillary lymphadenectomy: case report During lymphadenectomy in the left axilla of a 38-year-old woman with a 1.4 cm invasive ductal breast carcinoma, when the surgeon attempted to prepare the lateral margin of the pectoralis major muscle, an aberrant muscular slip was observed slightly lateral and deep to the pectoralis major muscle. The two muscles were separated by a narrow band of connective tissue that was completely dissected. Following this, the supernumerary muscle located in the center of the surgical field was pulled towards the latissimus dorsi muscle. As a result, the Breast Cancer Research Vol 11 Suppl 1 VIII Madrid Breast Cancer Conference S15 muscle formed an arcuate course and the lymphadenectomy was carried out troublesomely through a limited field. Nevertheless, the amount of the lymph node dissected was satisfactory (N: 0/25). Based on the anatomical characteristics of the muscle, it was recognized as a pectoralis quartus muscle. To our knowledge this is the first report of a pectoralis quartus muscle as a surgical finding. The surgeon should be aware of the possible presence of this supernumerary muscle as well as its anatomical characteristics in order to avoid any complications.

P17
Phase I study of combination therapy with weekly paclitaxel and cyclophosphatamide for advanced or recurrent breast cancer Objective Although anthracycline is a key agent in breast cancer treatment, there is a concern that it may cause cardiotoxicity. Recently, the usefulness of combined therapy with docetaxel and cyclophosphamide (C) was reported. Because paclitaxel (P) has different features, such as induction of apoptosis and anti-angiogenic activity on weekly administration, establishment of combination therapy of P/C is required. We initiated a phase I study to determine the maximum tolerated dose and the recommended dose (RD) of the combination therapy of P/C for advanced or recurrent breast cancer.
Methods P was given intravenously on days 1, 8 and 15 and C on day 1, every 3 weeks. P was given at 80 mg/m 2 for level 1 and 100 mg/m 2 for level 2, and C at 600 mg/m 2 for both. Onset of dose-limiting toxicity was evaluated during course 1, and tolerability through course 4. Results Four patients each were enrolled in levels 1 and 2 from October 2006 to November 2007. Main adverse events were four cases of grade 3 neutropenia (50%) and one case of peripheral nerve disorder (12.5%). During the first course of levels 1 and 2, hematologic toxicity of grade 4 and nonhematologic toxicity of grade 3 or higher were not observed, and a MTD was not attained. The response rate among assessable cases (one in level 1, two in level 2) was 66.7%. Conclusions Safety was confirmed during four courses at level 2, and this was regarded as the RD. This is the first report on the phase I study of the combination therapy of weekly P and C, and its safety and efficacy should be evaluated in the phase II trial. Introduction Secondary arm lymphoedema is a chronic and distressing condition, relatively common after axillary lymph node dissection (AND) for breast cancer. It may be associated with functional, esthetic, and psychological problems that actually could affect the quality-of-life (QOL) of breast cancer survivors. The present study describes the prevalence and characteristics of arm and hand swelling in patients undergoing breast cancer surgery in our institution.

Role of the BRCA1 gene in stem cells and treatment of mammary gland cancer
Methods We studied patients who underwent AND for breast cancer who had been diagnosed 4 years ago. We analyzed the prevalence of lymphoedema in these patients and reported data about pathologic and surgical characteristics.
Results Seventy-five women, median age 61 years, were included. Nine women (12%) reported arm or hand swelling since their surgery, eight of them with mild swelling. Twenty-one (28%) reported current swelling and one-half of them constant swelling, mainly in the hand. Swelling was considered mild when it affected just the hand. Women who reported severe swelling had significantly worse physical functioning and depressive symptoms as well. Since the swelling has reduced the possibility of dressing appropriately and modified perceptions about general appearance, 92% of these women began treatment for swelling with a high grade of compliance (86%). We detected that just five women had high body mass index; all of them had more than 10 axillary lymph nodes evaluated and one-half of them were affected by the tumor.
Conclusions Arm lymphoedema is a chronic problem for a subgroup of long-term survivors of breast cancer that negatively affects physical functioning. Educational efforts are needed for being aware of this problem so that survivors could identify this complication early and begin treatment to improve their physical functioning. Results Data are available for 40 patients. We found ≥1 CTCs before the first cycle of treatment with bevacizumab in 60% of the patients (n = 24). After the first treatment, reduction of one or more CTCs was found in 35% of the patients (n = 14). The median number of CTCs was 15 cells/7.5 ml blood in the first determination and 8 cells/7.5 ml in the second determination. In 30% of the patients (n = 12) we found an increase of baseline CTCs before and after treatment. In 26 patients we did not found any variation of baseline CTCs before and after treatment. In eight patients with CTCs positive at baseline (20%) the second determination after treatment was 0 cells/7.5 ml. Persistence of at least one CTC after the first cycle of treatment was found in 35% of the patients.

Conclusions
The results of this explorative study are preliminary and a large number of patients and follow-up are required. The study is ongoing to explore the prognostic significance of reduction or persistency of CTCs during treatment with anti-angiogenic agents (bevacizumab) and the relationship with response rate to treatment. Complete data will be presented. In the triple negative, the tumour size was pT0 0 (0%), pT1a 1 (7.1%), pT1b 1 (7.1%), pT1c 8 (57.1%), pT2 3 (21.4%), pT4b 1 (7.1%); and the axillary lymph node was pN0 9 (64.3%), pN1 5 (35.7%), with no statistically significant differences for the nontriple negative. The histological grade was grade III in 52.9% of the cases of triple-negative breast cancer and in 13.8% of the cases of nontriple-negative breast cancer; these differences were statistically significant. The overall survival was statistically worse, the local recurrences and contralateral breast cancer were higher in triple negative.

PROFFERED
Conclusions Triple-negative breast cancer has a high histological grade, more metastases, more local recurrences and contralateral breast cancer, and worse overall survival.

P27
Discordance between hormone receptor profile of primary breast cancer and metastatic bone disease: should bone marrow biopsy be considered a standard of care? Discordant receptor expression between primary and metastatic tumours has been reported in around 20 to 60% of cases. The present study, therefore, aimed to prospectively explore the incidence of discordant receptor status in primary and metastatic bone disease, and to evaluate the role of bone marrow biopsies for the reassessment of receptor status. Methods Nineteen patients with known bone metastases underwent both a CT-guided bone metastasis biopsy as well as bone marrow aspirate and trephine. The estrogen receptor (ER) and progesterone receptor (PR) of these samples was assessed and compared with those of primary breast cancer. Results Tumor cells were found in 13 (68.4%) bone metastasis samples and in nine (47.4%) bone marrow biopsies. Discordance between the primary and metastatic samples was seen in 10 patients (52.6%). Among these, ER and PR changed from positive to negative in seven cases and from negative to positive in one case. In six cases (31.6%), malignant cells were identified in both bone metastasis and bone marrow samples from the same patient. Among these, ER and PR were concordant in 100% and 83% of cases.
Conclusions Given that the receptor profile of metastatic disease is assumed to be the same as the primary tumour, discordance between primary and metastatic cancer can have a significant impact on the outcome of treatment choices. Receptor discordance rates in this analysis were similar to that which has been reported in previous studies. There appeared to be good concordance between bone metastasis and bone marrow biopsies. Bone marrow biopsy may therefore be a simple, safe and well-tolerated way to obtain tissue to reassess receptor status of metastatic breast cancer, and should be considered before the more invasive bone metastasis biopsy. Breast cancer is the leader of cancer deaths among women. Despite developments in surgery, chemotherapy and radiotherapy, the longterm survival of patients with metastatic and higher staging breast cancer has remained very low. Breast cancers and their aggressive nature are characterized by overexpression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR). There is a common downstream signaling molecule involved in both EGFR and VEGFR mediated pathways. There are reports that cancer cells acquired resistance to anti-EGF/EGFR therapy by increased tumor-induced angiogenesis due to the constitutive overexpression of VEGF. Hence, the inhibition of both EGFR and VEGFR signaling pathways simultaneously may provide greater benefit than blockade of either pathway alone. There are also reports that breast cancer acquired resistance to conventional chemotherapy and radiotherapy by increased phosphorylation of EGFR and VEGFR. Moreover, patients suffering from breast cancer have a poor prognosis because of a lack of effective treatment strategies. We hypothesized that inhibiting the phosphorylation of the EGFR and VEGFR by ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR, in combination with paclitaxel or radiation would inhibit breast cancer cell growth. We tested this hypothesis using the human breast cancer cell lines MCF-7 and MDA-MB-231. In culture, ZD6474 in combination with paclitaxel or UVB had a synergistic effect in inhibition of cell proliferation, and lowered the IC 50 by 10-fold and fivefold, respectively, compared with paclitaxel/UVB alone. Flow cytometry data suggest that combination therapy of ZD6474 with paclitaxel increases apoptosis from 20% to 45% in MDA-MB-231 and from 16% to 38% in MCF-7 compared with paclitaxel alone. This is further supported by a decrease in cell cycle regulatory protein cyclin D 1 and an increase in apoptosis marker poly(ADP-ribose) polymerase in combination therapy compared with paclitaxel and UVB irradiation alone. Moreover, combination therapy sensitizes the paclitaxel resistance MCF-7/PTX and MDA-MB-231/PTX. ZD6474 along with paclitaxel or radiation can therefore be used for treatment in drug-insensitive advanced breast cancers. program mandated prior therapy with tamoxifen and both steroidal and nonsteroidal aromatase inhibitors. Charts from the seven highest accruing centers were reviewed. The sample size was based on the derivation of a model to predict the probability of a patient remaining on fulvestrant and free from chemotherapy for at least 3 months. Results A total of 305 women received at least one dose of fulvestrant; 207 went on to receive chemotherapy (68%). Of these, 48 (23%) required chemotherapy at 3 months, 113 (55%) at 6 months, and 170 (82%) by 12 months. The median duration of fulvestrant treatment was 126 days (range 23 to 1,920). The median overall survival from start of fulvestrant was 698 days (25th percentile, 316 days to 75th percentile, 1,359 days). The preliminary prediction model showed that older age (OR = 0.96, 95% CI = 0.93 to 0.99) and having received no adjuvant hormonal therapy (OR = 0.5, 95% CI = 0.2 to 1.25) predicted a greater chance of remaining chemotherapyfree at 3 months. Presence of lung (OR = 2.55, 95% CI = 1.1 to 5.9) or brain metastases (OR = 12.8, 95% CI = 4.1 to 55.4) predicted a lower chance of remaining chemotherapy-free at 3 months.

P29
Conclusions Older age and having received no prior adjuvant hormonal therapy predicted a greater chance of remaining chemotherapy-free at 3 months, while lung and brain metastases predicted a lower chance. These factors may be considered when prescribing fulvestrant.

P30
Employing multiplex immunofluorescence to quantify Her2 and phosphorylated Her2 in formalin-fixed, paraffin-embedded breast tumor specimens Objective Her2 overexpression is a predictor for response to trastuzumab; however, only one-third of patients initially respond and the majority progress within 1 year. Recently, levels of the phosphorylated form of Her2 (pHer2) in hormone receptor-positive, primary tumors was determined to be an independent predictor for poor disease-free and overall survival, suggesting that additional variables may be important for determining outcome. We sought to develop a multiplex immunofluorescent (IF) quantitative assay for Her2 that included pHer2 and to compare the initial results with traditional methods of evaluating Her2 levels in formalin-fixed, paraffin-embedded (FFPE) breast tissue specimens.

Results
We developed baseline intensity thresholds from the component biomarkers in the Her2mplex (TAB250 Her2, c-erb B2 and phosphorylated Her2) utilizing a series of BTC: MCF7, T47D and SKBR3. IF images were acquired from 72 TMA cores, and 52 cores (26 patients) had appropriate tumor content (>50%) and quality for analysis. Both c-erB B2 and pHer2 were significantly associated with IHC data and were selected (AUC = 0.96, sensitivity = 0.91, specificity = 1) to discriminate Her2 (2+) from Her2 (3+). Conclusions Quantitation of Her2 and pHer2 by IF is feasible in FFPE breast tumor specimens. The results correlate with IHC protein data and also provide a quantitative strategy for the development of a standardized assay useful for tumor phenotyping, patient stratification and therapeutic indication.