New IR imaging modalities for cancer detection and for intra-cell chemical mapping with a sub-diffraction mid-IR s-SNOM.

We present two new modalities for generating chemical maps. Both are mid-IR based and aimed at the biomedical community, but they differ substantially in their technological readiness. The first, so-called "Digistain", is a technologically mature "locked down" way of acquiring diffraction-limited chemical images of human cancer biopsy tissue. Although it is less flexible than conventional methods of acquiring IR images, this is an intentional, and key, design feature. It allows it to be used, on a routine basis, by clinical personnel themselves. It is in the process of a full clinical evaluation and the philosophy behind the approach is discussed. The second modality is a very new, probe-based "s-SNOM", which we are developing in conjunction with a new family of tunable "Quantum Cascade Laser" (QCL) diode lasers. Although in its infancy, this instrument can already deliver ultra-detailed chemical images whose spatial resolutions beat the normal diffraction limit by a factor of ∼1000. This is easily enough to generate chemical maps of the insides of single cells for the first time, and a range of new possible scientific applications are explored.

An Approach to Breast Cancer Immunotherapy: The Apoptotic Activity of Recombinant Anti-Interleukin-6 Monoclonal Antibodies in Intact Tumour Microenvironment of Breast Carcinoma.

Current work is one of our comprehensive preclinical studies, a new approach to breast cancer (BC) immunotherapy through induction of tumour cell apoptosis. Tumour growth is not just a result of uncontrolled cell proliferation but also of reduced apoptosis. High levels of interleukin-6 (IL-6) are associated with metastatic BC and correlated with poor survival as it promotes growth of tumour-initiating cells during early tumorigenesis protecting these cells from apoptosis. Therefore, this study aims at investigating the potential of anti-IL-6 monoclonal antibodies to suppress IL-6 proliferative/anti-apoptotic activities in intact tumour microenvironment of BC. Fresh sterile tumour and normal breast tissue specimens were taken from 50 female Egyptian patients with BC undergoing radical mastectomy. A unique tissue culture system designed to provide cells of each intact tumour/normal tissue sample with its proper microenvironment either supplemented or not with anti-IL-6 monoclonal antibodies. To evaluate the apoptotic activity of anti-IL-6 as a novel candidate for BC treatment strategy, we compared its effects with those obtained using tumour necrosis-related apoptosis-inducing ligand TRAIL as an established apoptotic agent. Our results revealed that levels of either anti-IL-6- or TRAIL-induced apoptosis in the tumour or normal tissue cultures were significantly higher than those in their corresponding untreated ones (P < 0.001). No statistically significant differences have been found between apoptosis levels induced by anti-IL-6 monoclonal antibodies and those induced by TRAIL. Recombinant anti-IL-6 monoclonal antibodies could represent a novel effective element of immunotherapeutic treatment strategy for BC. The selectivity and anti-apoptotic potential of anti-IL-6 is highly hopeful in IL-6- abundant BC tumour microenvironment.

Angiotensin II up-regulates monocarboxylate transporters expression in the rat adrenal gland.

Angiotensin II (Ang II) is a major regulator of aldosterone secretion in the adrenal zona glomerulosa because it up-regulates the expression of a large number of genes involved in aldosterone biosynthesis. The transport of acetate across adrenocortical cells is a crucial step in the de novo synthesis of cholesterol, the steroid precursor of aldosterone. However, whether Ang II can affect this transport remains unknown. The current study aims to investigate the effect of in vivo infusion of Ang II on monocarboxylate transporters (MCT1, MCT2, and MCT4) gene expression in the rat adrenal gland. Immunohistochemical analysis and real-time PCR were used to examine the expression of MCTs at the protein and mRNA levels, respectively. The immunohistochemical analysis showed that higher numbers of cells expressed MCT1, MCT2, and MCT4 proteins in the zona glomerulosa and zona fasiculata of the adrenal cortex of Ang II-infused rats. Furthermore, real-time PCR indicated that in vivo infusion of Ang II increased the mRNA levels of MCT1, MCT2, and MCT4 in the rat adrenal gland. MCT up-regulation might maximize the intracellular transport of acetate in response to the stimulatory effect of Ang II on aldosterone secretion by the adrenal zona glomerulosa..

Lesions of uncertain malignant potential in the breast (B3): what do we know?

Breast lesions classified as of uncertain malignant potential (B3) on biopsy form a diverse group of abnormalities, which pose a diagnostic and management challenge. In this paper, we discuss the imaging and pathology features as well as the management of the most controversial B3 lesions, consisting of papillary lesions, complex sclerosing lesions/radial scars, lobular intraepithelial neoplasia, and atypical epithelial proliferation of ductal type. As there is an association with malignancy at the time of diagnosis, as well as an increase in the risk of subsequent development of cancer, a multidisciplinary discussion is almost always required to tailor treatment.

Central neuropeptide W has anorexigenic effect in rats.

Neuropeptide W (NPW) is produced in neurons located in hypothalamus, brain stem and antral G cells and its receptors are present in the hypothalamus, in particular in the paraventricular nucleus (PVN). There are two forms of the peptide, designated as neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). Neuropeptide W is an endogenous ligand for G-protein-coupled receptor, GPR7 and GPR8 receptors (R), which in humans are expressed in the hypothalamus and probably involved in the control of energy homoeostasis and neuroendocrine axes. We conducted this study to investigate the effects of NPW on feeding intake and energy expenditure in Wistar rats. Systemic (icv) injection of both forms of neuropeptide W (NPW23 and NPW30) to ad libitum feeding Wistar rats decreased dark feeding and fasting-induced feeding. One week of systemic treatment with NPW23 decreased feeding intake and weight gain during the treatment period. On the other hand, systemic treatment with antineuropeptide W antibody increased feeding intake. Moreover, systemic treatment with neuropeptide W-23 raised body temperature and consequently thermogenesis. These results strongly suggest that neuropeptide W may play an important central role in the feeding intake and energy balance control in mammals.

An open-label study of lapatinib in women with HER-2-negative early breast cancer: the lapatinib pre-surgical study (LPS study).

BACKGROUND: This phase II, open-label, multicentre study aimed to evaluate changes in cell proliferation and biomarkers, as well as efficacy of lapatinib in treatment-naïve patients with HER-2-negative primary breast cancer. PATIENTS AND METHODS: Patients received 1500 mg lapatinib for 28-42 days before surgery with repeat biopsies and measurements. The primary end point was inhibition of cell proliferation measured by Ki67; the secondary end points included clinical response, adverse events and changes in FOXO3a, FOXM1, p-AKT and HER-3. RESULTS: Overall, there was no significant reduction in Ki67 with treatment (assessment carried out in 28 of 31 subjects enrolled). However, four patients (14%) showed a reduction in Ki67 ≥50%. Four of 25 patients (16%) had a partial response to treatment judged by sequential ultrasound measurements. Response, in terms of either Ki67 or ultrasound, did not relate to changes in any biomarker assessed at baseline, including the estrogen receptor (ER) and epidermal growth factor receptor (EGFR). However, all four clinical responders were HER-3 positive, as were three of four Ki67 responders. CONCLUSIONS: Overall, a pre-surgical course of lapatinib monotherapy had little effect on this group of patients; however, in subsets of patients, especially those with HER-3-positive tumors, we observed either reduction in proliferation (Ki67) or tumor size; EGFR/ER status had no impact.

Multicentre evaluation of intraoperative molecular analysis of sentinel lymph nodes in breast carcinoma.

BACKGROUND: Ideally, intraoperative sentinel lymph node (SLN) analysis in breast cancer should be automated, have high concordance with extensive histopathology, and be applicable in any hospital setting. A prospective multicentre evaluation of the one-step nucleic acid amplification (OSNA) automated molecular diagnostic system of SLN analysis was undertaken. METHODS: Intraoperative examination of SLNs from 204 patients with breast cancer was performed by OSNA at four sites in the UK. Half of each SLN was assessed by OSNA (for cytokeratin 19 mRNA) and the remaining half was paraffin embedded for intensive histological examination at ten levels. Discordant cases were reanalysed by further molecular biological techniques and by additional histological examination of all remaining nodal material to ascertain whether the discordance was due to an uneven distribution of metastases, known as tissue allocation bias (TAB). RESULTS: After exclusion of samples affected by TAB, the overall concordance rate for OSNA versus histopathology was 96.0 per cent, with a sensitivity of 91.7 per cent and a specificity of 96·9 per cent. The median time to process a single SLN was 32 (range 22-97) min, and that for two nodes 42 (30-73) min. CONCLUSION: OSNA enables accurate automated intraoperative diagnosis and can be used successfully in different UK hospitals. When the SLN is shown to be positive, the patient can undergo immediate axillary clearance under the same anaesthetic rather than having a delayed second procedure.

The DEAD-box protein p72 regulates ERalpha-/oestrogen-dependent transcription and cell growth, and is associated with improved survival in ERalpha-positive breast cancer.

The DEAD-box RNA helicases p68 (DDX5) and p72 (DDX17) have been shown to act as transcriptional co-activators for a diverse range of transcription factors, including oestrogen receptor-alpha (ERalpha). Here, we show that, although both proteins interact with and co-activate ERalpha in reporter gene assays, small interfering RNA-mediated knockdown of p72, but not p68, results in a significant inhibition of oestrogen-dependent transcription of endogenous ERalpha-responsive genes and oestrogen-dependent growth of MCF-7 and ZR75-1 breast cancer cells. Furthermore, immunohistochemical staining of ERalpha-positive primary breast cancers for p68 and p72 indicate that p72 expression is associated with an increased period of relapse-free and overall survival (P=0.006 and 0.016, respectively), as well as being inversely associated with Her2 expression (P=0.008). Conversely, p68 shows no association with relapse-free period, or overall survival, but it is associated with an increased expression of Her2 (P=0.001), AIB-1 (P<0.001) and higher tumour grade (P=0.044). Our data thus highlight a crucial role for p72 in ERalpha co-activation and oestrogen-dependent cell growth and provide evidence in support of distinct but important roles for both p68 and p72 in regulating ERalpha activity in breast cancer.

Evaluation of automated silver-enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens.

AIMS: To validate the use of the silver-enhanced in situ hybridization (SISH) technique in assessing HER2 status of breast carcinoma in excision biopsy specimens, and to assess its reliability in determining HER2 status in core biopsy specimens. METHODS AND RESULTS: Routinely processed paraffin sections of 65 excised breast carcinomas and 56 available preoperative core biopsy specimens from the same patients were selected from the archives for testing with the SISH technique using the automated Ventana Benchmark XT machine. For each case, two sections were used, one for the assessment of HER2 gene amplification and the other for assessment of chromosome 17. Of the 65 excision specimens tested, sections of 53 cases were also available for fluorescence in situ hybridization (FISH) examination. HER2 gene amplification was detected by SISH in 14 (21%) out of 65 excision specimens and in eight (14%) out of 56 core biopsy specimens. The results of SISH and FISH were identical in 50 (94%) out of the 53 excision cases examined by the two techniques. Two cases were SISH-, FISH+, and one case was the other way round. SISH results of core biopsy specimens and corresponding excision biopsy specimens were identical in 50 (89%) out of 56 cases. Four cases (7%) were SISH- in cores but positive in excision specimens, whereas two cases were the other way round. CONCLUSIONS: The results validate the use of the SISH technique for assessing HER2 status of excised breast carcinoma tissue sections. The results are comparable to those obtained with FISH, but SISH has the advantage of having a permanent end result that can be visualized by an ordinary light microscope. There is a reasonable 89% concordance between SISH results obtained in core and excision biopsy specimens. However, it may be prudent to postpone doing SISH, if possible, until sections of the resected specimen are available, as these seem to be more reliable.

Colorectal breast metastases presenting with atypical imaging features.

Breast metastases from non-breast primaries are rare in female patients and exceedingly rare in male patients, with only a handful of cases described. Lymphoma, metastatic melanoma and bronchial carcinoma are the primary sites for the majority of breast metastases. Breast metastases from colorectal carcinoma have been described previously in only a small number of cases in the literature. Here, we report a further two patients with biopsy-proven colorectal carcinoma metastases to both breasts, who demonstrate contrasting unusual and atypical imaging features that have not been reported previously. In one case, the imaging appearances mimic a multifocal primary breast carcinoma. Metastatic disease in the breast is a marker for disseminated metastatic spread, with a correspondingly poor prognosis. Therefore, we review the imaging features that differentiate metastatic breast disease from multifocal breast primaries, which are important to recognize because the management options for these patients differ greatly.

Neoadjuvant 5-fluorouracil, epirubicin and cyclophosphamide chemotherapy followed by docetaxel in refractory patients with locally advanced breast cancer.

The objective of this study was to evaluate the clinical response of locally advanced breast cancer (LABC) to neoadjuvant (NA) chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) and to study the role of docetaxel in patients who fail to respond to first-line chemotherapy. Patients were enrolled who had primary tumours without distant metastasis that were too extensive for conservative surgery. All underwent NA chemotherapy for breast cancer and thereafter surgery and/or radical radiotherapy. NA chemotherapy with FEC was administered to 88 patients between February 1998 and June 2005. A median of 6 cycles of FEC (range 1-8) was given, followed in 21 cases by a median of 4 cycles (range 2-6) of docetaxel. Where clinically established, with FEC the clinical complete response (cCR) was 22/81 (27%), clinical partial response (cPR) 41/81 (51%), clinical stable disease (cSD) 18/81 (22%). In patients where the response to FEC was regarded as insufficient, docetaxel was given. Response rates were cCR 3/21 (14%); cPR 10/21 (48%), cSD 8/21 (38%). There were 11 cases of pathological complete response (pCR), 9 in the FEC-only group and 2 in the docetaxel group. Following chemotherapy 49 (56%) patients underwent mastectomy, 32 (36%) breast conserving surgery and 5 (6%) radical radiotherapy, giving a breast conservation rate of 42%. Two patients died before receiving surgery or radical radiotherapy. The results show that neoadjuvant FEC is a reasonable NA therapy in breast cancer and that docetaxel is effective in FEC refractory cases. Only 8 of 81 (10%) assessable patients did not respond to any chemotherapy, giving an overall clinical response rate of 90%, which is comparable to studies in which taxanes were given irrespective of response to preceding therapy with antracycline including regimes.

Phosphorylation of ERalpha at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERalpha phosphorylation in breast cancer progression.

Oestrogen receptor-alpha (ERalpha) is an important prognostic marker in breast cancer and endocrine therapies are designed to inhibit or prevent ERalpha activity. In vitro studies have indicated that phosphorylation of ERalpha, in particular on serine 118 (S118), can result in activation in a ligand-independent manner, thereby potentially contributing to resistance to endocrine agents, such as tamoxifen and aromatase inhibitors. Here we report the immunohistochemistry (IHC) of S118 phosphorylation in 301 primary breast tumour biopsies. Surprisingly, this analysis shows that S118 phosphorylation is higher in more differentiated tumours, suggesting that phosphorylation at this site is associated with a good prognosis in patients not previously treated with endocrine agents. However, we also report that S118 phosphorylation was elevated in tumour biopsies taken from patients who had relapsed following tamoxifen treatment, when compared to pre-treatment biopsies. Taken together, these data are consistent with the view that S118 phosphorylation is a feature of normal ERalpha function and that increases in levels of phosphorylation at this site may play a key role in the emergence of endocrine resistance in breast cancer.

Intra-operative assessment of excision margins using breast imprint and scrape cytology.

Local recurrence in breast cancer surgery is related to the completeness of excision. Histological analysis of excision margins is time consuming and impractical for use intra-operatively. Our group evaluated breast imprint and scrape cytology (ISC) for the assessment of excision margins in a feasibility study in 1993-4, with 10 year clinical follow-up. Twenty-six consecutive women undergoing 27 wide local excisions for breast cancer had excision margins prospectively assessed with intra-operative ISC blinded to histology. All ISC results were ready (range 22-30 min) before surgery was completed. ISC agreed with histology in 21/27 (=78%) and disagreed in 6/27 (=22%) of the cases. In two cases with local recurrence, histology was positive in one case, whereas ISC margins were positive in both. Intra-operative ISC is reliable and could help the surgeon to excise more tissue to prevent a second (re-excision) operation. ISC margins may predict clinical outcome, although a larger interventional follow-up study is required.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF.

Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERalpha plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERalpha and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERalpha is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

CD10 immunostaining of bile canaliculi in liver biopsies: change of staining pattern with the development of cirrhosis.

AIMS: This study was prompted by published observations concerning the absence of normal bile canalicular CD10 staining in some cases of primary liver cell carcinoma. Our aim was to investigate the possibility that this loss of staining occurs prior to the development of cancer. METHODS AND RESULTS: The study comprised 164 liver biopsies, including 96 from patients with hepatitis C infection of various grades and stages including nine cases with cirrhosis, 24 other cases of cirrhosis of other aetiology, five cases of primary liver carcinoma, 12 cases of metastatic carcinoma, as well as biopsies with a variety of other liver diseases. CD10 was demonstrated in paraffin sections using the avidin-biotin immunoperoxidase technique. In hepatitis C cases, a significant loss of the canalicular pattern was seen in four out of 41 (10%) biopsies with stages 0-1 compared with 25 out of 55 (45%) with stages 2-6 (P < 0.001). There was also a significant difference (P < 0.001) between biopsies with stage 2-3 and those with stage 4-6, where marked pattern loss was seen in 9/37 (24%) and 16/18 (89%), respectively. Marked loss of the pattern was also seen in 16 out of the 24 (67%) other cirrhotic biopsies, as well as in cases with severe lobular inflammation and cholestasis and liver cell dysplasia and carcinoma. In hepatitis C biopsies, no relationship was noted between the reduction in the canalicular pattern and the necroinflammatory score. CONCLUSIONS: CD10-stained bile canalicular pattern in liver biopsies is preserved in cases with mild fibrosis and inflammation, but it becomes increasingly reduced with the advance of fibrosis or the presence of severe lobular inflammation or extensive metastases. Further investigations into the relationship between the changes in CD10 staining pattern and liver function tests may be useful in explaining test results.