HER2-HER3 dimer quantification by FLIM-FRET predicts breast cancer metastatic relapse independently of HER2 IHC status.

Overexpression of HER2 is an important prognostic marker, and the only predictive biomarker of response to HER2-targeted therapies in invasive breast cancer. HER2-HER3 dimer has been shown to drive proliferation and tumor progression, and targeting of this dimer with pertuzumab alongside chemotherapy and trastuzumab, has shown significant clinical utility. The purpose of this study was to accurately quantify HER2-HER3 dimerisation in formalin fixed paraffin embedded (FFPE) breast cancer tissue as a novel prognostic biomarker.FFPE tissues were obtained from patients included in the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) study. HER2-HER3 dimerisation was quantified using an improved fluorescence lifetime imaging microscopy (FLIM) histology-based analysis. Analysis of 131 tissue microarray cores demonstrated that the extent of HER2-HER3 dimer formation as measured by Förster Resonance Energy Transfer (FRET) determined through FLIM predicts the likelihood of metastatic relapse up to 10 years after surgery (hazard ratio 3.91 (1.61-9.5), p = 0.003) independently of HER2 expression, in a multivariate model. Interestingly there was no correlation between the level of HER2 protein expressed and HER2-HER3 heterodimer formation. We used a mathematical model that takes into account the complex interactions in a network of all four HER proteins to explain this counterintuitive finding.Future utility of this technique may highlight a group of patients who do not overexpress HER2 protein but are nevertheless dependent on the HER2-HER3 heterodimer as driver of proliferation. This assay could, if validated in a group of patients treated with, for instance pertuzumab, be used as a predictive biomarker to predict for response to such targeted therapies.

Models of breast morphogenesis based on localization of stem cells in the developing mammary lobule.

Characterization of normal breast stem cells is important for understanding their role in breast development and in breast cancer. However, the identity of these cells is a subject of controversy and their localization in the breast epithelium is not known. In this study, we utilized a novel approach to analyze the morphogenesis of mammary lobules, by combining one-dimensional theoretical models and computer-generated 3D fractals. Comparing predictions of these models with immunohistochemical analysis of tissue sections for candidate stem cell markers, we defined distinct areas where stem cells reside in the mammary lobule. An increased representation of stem cells was found in smaller, less developed lobules compared to larger, more mature lobules, with marked differences in the gland of nulliparous versus parous women and that of BRCA1/2 mutation carriers versus non-carriers.

Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium.

INTRODUCTION: Although estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER-, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes. METHODS: We used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER- mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells. RESULTS: We describe a cellular hierarchy in the normal human mammary gland in which ER-/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis. CONCLUSIONS: This study presents direct evidence that ER+ cells are generated by ER-/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and clonogenicity in suspension culture. This latter effect is reversed by treatment with retinoic acid.

Classification of terahertz-pulsed imaging data from excised breast tissue.

We investigate the efficacy of using data reduction techniques to aid classification of terahertz (THz) pulse data obtained from tumor and normal breast tissue. Fifty-one samples were studied from patients undergoing breast surgery at Addenbrooke's Hospital in Cambridge and Guy's Hospital in London. Three methods of data reduction were used: ten heuristic parameters, principal components of the pulses, and principal components of the ten parameter space. Classification was performed using the support vector machine approach with a radial basis function. The best classification accuracy, when using all ten components, came from using the principal components on the pulses and principal components on the parameter, with an accuracy of 92%. When less than ten components were used, the principal components on the parameter space outperformed the other methods. As a visual demonstration of the classification technique, we apply the data reduction/classification to several example images and demonstrate that, aside from some interpatient variability and edge effects, the algorithm gives good classification on terahertz data from breast tissue. The results indicate that under controlled conditions data reduction and SVM classification can be used with good accuracy to classify tumor and normal breast tissue.

Terahertz pulsed spectroscopy of freshly excised human breast cancer.

The complex refractive indices of freshly excised healthy breast tissue and breast cancers collected from 20 patients were measured in the range of 0.15 - 2.0 THz using a portable terahertz pulsed transmission spectrometer. Histology was performed to classify the tissue samples as healthy adipose tissue, healthy fibrous breast tissue, or breast cancers. The average complex refractive index was determined for each group and it was found that samples containing cancer had a higher refractive index and absorption coefficient. The terahertz properties of the tissues were also used to simulate the impulse response functions expected when imaging breast tissue in a reflection geometry as in terahertz pulsed imaging (TPI). Our results indicate that both TPS and TPI can be used to distinguish between healthy adipose breast tissue, healthy fibrous breast tissue and breast cancer due to the differences in the fundamental optical properties.

Lymphatic drainage pathways of the breast and the upper limb.

OBJECTIVE: To determine how often the sentinel lymph node (SLN) draining the breast is the same node as the SLN draining the upper limb. A common SLN might increase the risk of upper limb breast cancer-related lymphoedema after SLN biopsy. METHODS: Patients with invasive breast cancer, identified as being suitable for axillary lymph node dissection, were injected preoperatively with 40 MBq of technetium-99m (Tc)-human polyclonal immunoglobulin G intradermally into the ipsilateral breast and 3 MBq of indium-111 (In)-human polyclonal immunoglobulin G intradermally into the ipsilateral hand, or vice versa. Axillary lymph nodes were removed, separated and assayed in a well counter for Tc and In. RESULTS: Fifteen patients entered the study. In 13 of 15 patients, the 'hottest' lymph node for Tc was separate from the 'hottest' lymph node for In. In two of 15 patients the 'hottest' lymph node for Tc was also the 'hottest' lymph node for In, suggesting a common drainage pathway from the ipsilateral breast and upper limb. CONCLUSION: Although the majority of patients has different pathways of lymphatic drainage from the ipsilateral breast and upper limb, in a small minority of patients the drainage pathway is through a common SLN. Such patients may be at increased risk of developing upper limb breast cancer-related lymphoedema after SLN biopsy.

Lymphatic drainage in the muscle and subcutis of the arm after breast cancer treatment.

Breast cancer-related lymphoedema of the arm (BCRL) results from impaired lymph drainage after axillary surgery. Little is known about lymphatic changes in the arm between surgery and oedema onset. We measured forearm muscle and subcutis lymph drainage in 36 women at 7 and 30 months after surgery by quantitative lymphoscintigraphy. None had BCRL initially but 19% had BCRL by 30 months. At 7 months muscle and subcutis drainage in both arms of BCRL-destined women exceeded that of non-BCRL women (P < 0.01). Muscle lymph drainage always exceeded subcutis drainage (P < 0.0001). Muscle lymph drainage in the ipsilateral arm was unimpaired relative to the contralateral arm. BCRL therefore developed in women with higher peripheral lymph flows. The major lymphatic load was generated by muscle; there was no pre-BCRL lymphatic impairment in the muscle of the ipsilateral arm. We propose that some women have a defined, constitutive predisposition to secondary lymphoedema. Specifically, women with higher filtration rates, and therefore higher lymph flows through the axilla that are closer to the maximum sustainable, are at greater risk of BCRL following axillary trauma, even following removal of 1-2 nodes.

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 alpha6-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 alpha6-integrin as a potential therapeutic target aimed at tumour-generating subsets of breast cancer cells.

Breast cancers: noninvasive method of preoperative localization with three-dimensional US and surface contour mapping.

Formal ethical approval was granted by the local research ethics committee; all participants gave written consent. The purpose of the study was to prospectively evaluate the feasibility of a noninvasive method of breast tumor localization in 25 participants, based on the coregistration of three-dimensional (3D) ultrasonographic (US) data with surface contour data obtained by using a 3D laser camera. The tumor is segmented from the US data, and a surface-rendered 3D image of the tumor, in relation to the breast surface contour, is produced. From a personal computer in the operating room, the surgeon can dynamically view a 3D image of the tumor within the breast. This noninvasive method was equivalent to conventional techniques in 18 of 25 patients but was less successful in larger-breasted patients. In selected patients, this localization method could provide an alternative to conventional invasive techniques and can offer both spatial localization and tumor morphology.

High definition three-dimensional ultrasound to localise the tumour bed: a breast radiotherapy planning study.

BACKGROUND AND PURPOSE: Complex radiation techniques, such as conformal radiotherapy for partial breast irradiation, require accurate localisation of the tumour bed. This study investigated high definition 3D ultrasound for breast tumour bed localisation. Study aims were: firstly, to determine how easily a tumour cavity could be visualised with 3D ultrasound; secondly, to determine the accuracy of computed tomography (CT) and 3D ultrasound co-registration; thirdly, to compare 3D ultrasound with other methods of localisation. MATERIALS AND METHODS: 3D ultrasound examinations were carried out in 40 women attending for breast radiotherapy. 3D position data were co-registered with the radiotherapy planning CT. 2D ultrasound and CT, surgical clips and CT, and CT alone were also used to localise the tumour bed in 32/40, 14/40 and 5/40 patients, respectively. Tumour bed volume and centre of gravity measurements for all methods of localisation were compared. RESULTS: Mean surgery to imaging interval was 44 days (range 23-86 days). The post-operative cavity was seen in all cases using the 3D ultrasound, and was graded as highly visible, visible and subtle in 21/40 (53%), 12/40 (30%) and 7/40 (17%) cases, respectively. There was a statistically significant improvement in the ability of 3D ultrasound to localise the tumour bed compared with 2D ultrasound. CT-ultrasound registration was achieved in all cases. Two-dimensional and 3D ultrasound showed smaller tumour bed volumes than clips. CONCLUSIONS: Three-dimensional ultrasound localisation of the tumour bed appears superior to 2D ultrasound. It can also be co-registered with a planning CT, thus allowing additional information on the size and location of the tumour bed to be integrated into complex radiotherapy planning.

Lymph node status and breast cancer-related lymphedema.

OBJECTIVE: This study examines the association between nodal positivity and risk of developing breast cancer-related lymphedema (BCRL) in patients who underwent axillary lymph node dissection (ALND). SUMMARY BACKGROUND DATA: The pathophysiology of BCRL is poorly understood. It has been assumed that one of the factors predisposing to the development of BCRL is nodal positivity, although retrospective series have produced contradictory findings. As these studies have included treatment regimens known to cause BCRL, such as axillary radiotherapy, any relationship between nodal positivity and the development of BCRL remains speculative. METHODS: A total of 212 patients who had undergone ALND for invasive breast cancer had arm volume measurements preoperatively, and at intervals postoperatively. No patient received axillary radiotherapy. Arm volumes were obtained by measuring serial arm circumferences every 4 cm up the arm and then calculated by using the formula for the volume of a truncated cone. Robust regression techniques were used to analyze the effects of node positivity, age, preoperative body mass index, and wound infection on arm volume excess. RESULTS: In all, 64 of 212 (30%) patients were node positive. Contrary to previous assumptions, positive node status was significantly inversely associated with arm volume after adjusting for tumor size, time since operation, and allowing for correlated observations within subjects. Furthermore, the number of positive nodes also correlated inversely with arm volume. CONCLUSION: These results are counterintuitive to the conventional understanding of the pathophysiology of BCRL. A possible explanation is that patients who develop disease in axillary lymph nodes and subsequently undergo ALND have more time and ability to develop lymphatic collaterals, which may provide adequate lymphatic drainage following surgery, thereby reducing the risk of developing BCRL.

Breast cancer, stem cells and prospects for therapy.

The mammary epithelium contains multipotent stem cells that give rise to all differentiated cell types present within the tissue. Mammary epithelial stem cells have been prospectively purified from dissociated mammary epithelium on the basis of cell surface antigen expression. It has become apparent in recent years that for breast cancer and other malignancies only a small proportion of tumour cells--'cancer stem cells'--have the capacity for extensive proliferation and transferral of the tumour. We review the evidence for breast cancer stem cells, we consider their relationship to mammary epithelial stem cells and we examine the implications for current and future therapeutic strategies.

Terahertz pulsed imaging of human breast tumors.

The feasibility of using terahertz pulsed imaging to map margins of exposed breast tumors was investigated by imaging 22 excised human breast tissue specimens with carcinoma excised from 22 women (mean age, 59 years; range, 39-80 years). The study was approved by the local ethics research committee, and informed consent was obtained from all patients. The size and shape of tumor regions on terahertz images were compared with those identified at histopathologic examination of the imaged section. Two image parameters were investigated: the minimum of the terahertz impulse function and the ratio of the minimum to the maximum of the terahertz impulse function. The correlation coefficient for the tumor area on images compared with that on a photomicrograph of all 22 samples was greater than 0.82 for both parameters. The shape of the tumor regions on terahertz images also correlated well with that on a photomicrograph (median Spearman rank correlation coefficient, 0.69). Findings of this study demonstrate the potential of terahertz pulsed imaging to depict both invasive breast carcinoma and ductal carcinoma in situ under controlled conditions and encourage further studies to determine the sensitivity and specificity of the technique.

Short-term effects of axillary lymph node clearance surgery on lymphatic physiology of the arm in breast cancer.

It is not known why some women develop breast cancer-related lymphedema (BCRL) of the arm, whereas others having similar treatment do not. We speculated that increased uptake of protein into local blood may protect against BCRL. Sixteen women were given bilateral subcutaneous hand webspace injections of polyclonal immunoglobulin (HIgG), (99m)Tc-HIgG on one side and (111)In-HIgG on the other, before and 3 mo after axillary clearance surgery. The rates of clearance of activity from the depot (k) and accumulation in central blood (b(contra)) were measured using a scintillation probe and bilateral antecubital vein blood sampling, respectively. Activity accumulating in blood ipsilateral to the injected side, in excess of central blood activity (b(ipsi)) was also calculated as a measure of local vascular uptake. The k correlated with b(contra), but neither changed in response to surgery. However, b(ipsi) for injections of (99m)Tc-HIgG into the affected arm increased in all seven patients in whom data were available (0.018 +/- 0.006 to 0.038 +/- 0.007%/min; P < 0.05); indeed, in five of these seven, b(ipsi) paradoxically exceeded b(contra), and none developed BCRL at 3-yr follow-up. We conclude that uptake of protein into local blood and/or proteolysis increases after axillary surgery and may protect against BCRL.

A 1-year prospective study of individual variation in distress, and illness perceptions, after treatment for breast cancer.

OBJECTIVE: The primary objective was to conduct a detailed analysis of individual variation in psychological morbidity in the year following surgery for breast cancer. The salience of the patients' "illness perceptions" to morbidity was examined as a secondary objective. METHODS: Psychological morbidity was assessed with the General Health Questionnaire (GHQ-28) in a prospective study of 371 women having surgery for primary breast cancer. Patients also completed the Illness Perception Questionnaire (IPQ), Mental Adjustment to Cancer Scale (MAC) and the Eysenck Personality Scales (EPS). Assessments were made postoperatively and at 3, 6 and 12 months after surgery. RESULTS: Whilst descriptive statistics indicated a general reduction in mean distress over the 12-month follow-up, close analysis showed that a quarter of all patients maintained clinically significant levels of distress throughout the period. Patients with chronically elevated distress were characterised by higher levels of neuroticism, greater symptom awareness, more pain and poorer self-rated general health. In the regression analysis, psychological morbidity across the 1-year follow-up was predicted principally by the immediate postoperative state of distress, IPQ symptom awareness and the perceived time line of the illness, general health and, to a more minor extent, by neuroticism. CONCLUSION: There is marked individual variation in psychological morbidity in the year following breast cancer surgery, which is reliably predicted by the patient's immediate postoperative state of distress, her perception of the impact of the symptoms and the time line of the disease. Subgroups of patients with chronically high distress are characterised by factors including personality and negative perceptions and beliefs about their illness.

The diagnosis and management of pre-invasive breast disease: problems associated with management of pre-invasive lesions.

The treatment of ductal carcinoma in situ (DCIS) involves adequate surgical excision with adjuvant radiotherapy where appropriate. An inadequate excision margin and young age are independent risk factors for local recurrence. Routine surgery to axillary lymph nodes is not recommended in pure DCIS. In localised DCIS, adjuvant radiotherapy is recommended on the basis of tumour size, margin width and pathological subtypes. The role of adjuvant tamoxifen as systemic therapy is controversial. The treatment of atypical ductal/lobular hyperplasia and lobular carcinoma in situ involves surgical excision to exclude coexisting DCIS or invasive disease.