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.

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.

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.

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.

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.

Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction.

Breast cancer micrometastases in axillary lymph nodes have been detected by serial sectioning and immunohistochemistry, and shown to have prognostic significance. We have used polymerase chain reaction (PCR) to see whether we could further improve the detection rate of micrometastases. Fifty-seven axillary lymph nodes from patients with breast cancer were examined histologically to assess the proportion of tumor involvement. Immunohistochemical staining with the use of an anti-keratin 19 antibody confirmed the histological findings. Reverse transcription PCR was then performed on extracted RNA by using K19 primers, and all 18 histologically involved nodes yielded the expected 460-base pair product. Of 39 histologically negative nodes, 4 (10%) gave K19 bands detectable with ethidium staining and a further 10 (28%) gave K19 bands after Southern hybridization. To further increase the detection sensitivity a two stage amplification was performed by using nested primers, and K19 product was found in lymph nodes from patients without cancer, as well as in all the nodes from cancer patients. This was shown to be genuine low level expression from endogenous mRNA template, and not derived from amplification of a K19 pseudogene. Reducing the number of PCR cycles in the two amplification steps did not allow sufficient discrimination between normal nodes and those involved nodes in which K19 expression was only detectable after Southern hybridization. The optimal "cut-off" point to distinguish involved nodes from normal nodes remained at the level of 40 cycles of PCR and Southern hybridization. PCR, using K19 as a tumor marker, has been demonstrated in this study to improve the detection of micrometastases in axillary lymph nodes in patients with breast cancer: sensitivity is limited by the specificity of the tumor marker.

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.

Mammary duct ectasia and pituitary adenomas.

Mammary duct ectasia developed in three postmenopausal patients who had had pituitary chromophobe adenomas. The first patient had bilateral duct ectasia that developed 8 and 11 years after hypophysectomy. The second patient, who also had bilateral ectasia, had a prolactin-producing pituitary adenoma for which bromocriptine was prescribed. The ectasia developed in one breast before commencing bromocriptine therapy, and in the other breast 2 years later. The third patient also had a prolactin-producing pituitary adenoma. Unilateral duct ectasia developed while bromocriptine was taken. The ectasia in all patients was very marked and affected all excised ducts. Cholesterol granulomas were sometimes very extensive. These cases suggest a relationship between certain hypothalamic/pituitary disorders, possibly related to prolactin secretion and the development of mammary duct ectasia in postmenopausal patients.

Presence of epstein-barr-virus in breast-carcinoma.

Epstein-Barr virus (EBV) is associated with human B cell lymphomas, but is also commonly found in the epithelial cells of undifferentiated nasopharyngeal carcinoma. As recent reports have also suggested a wider distribution particularly in lymphoepithelioma-like carcinomas, we investigated its presence in breast carcinomas of varying histology. Using PCR amplification of DNA extracted from paraffin sections, we found that EBV was present in 15/28 breast cancers. It seemed to be more prevalent in Paget's disease and in medullary carcinomas and less frequent in the invasive ductal cancers that constitute about 75% of breast carcinomas. This was confirmed by analysis of purified DNA extracted from a further 48 invasive ductal carcinomas: 19 were EBV positive. Latent membrane viral protein was detected by immunohistology in scattered epithelial tumour cells. The vast majority of lymphoid cells were unstained. A rough approximation indicates that there could be up to several hundred copies of the EBV genome per mu g of extracted tissue DNA. We were unable to demonstrate the presence of RNA by in situ hybridisation using fluorescein labeled EBER oligonucleotides but this may be due to technical reasons. No EBV DNA was found using PCR in the 12 normal breast specimens examined. These initial results suggest that the EBV genome is present in certain breast epithelial malignancies.

Secondary amyloidosis of the gastrointestinal tract: an electron microscopic study.

Biopsy specimens of the gastric antrum, duodenum, and rectum from three patients with secondary amyloidosis were examined by electron microscopy in an attempt to determine the ultrastructural distribution of amyloid filaments and to identify any secondary changes in the covering mucosal epithelial cells. The characteristic amyloid filaments were seen in the walls of submucosal arterioles and mucosal capillaries deposited within the basal lamina surrounding the endothelial cells. Filaments were also sometimes seen within the muscularis mucosa. the overlying gastric and rectal epithelial cells appeared normal, but numerous curved bacilli were seen in close contact with the microvilli of the surface epithelial gastric cells. Duodenal columnar absorptive cells were vacuolated and contained prominent lysosomes. These changes are probably degenerative and may explain, at least in part, the development of malabsorption in some patients with intestinal amyloidosis.

Alveolar variant of invasive lobular carcinoma of the breast. A tumor rich in estrogen receptors.

During a study of the correlation between the histologic types of breast carcinoma and their hormone receptor contents, eight cases of the alveolar variant of invasive lobular carcinoma were identified, and all had estrogen receptor protein concentrations higher than 400 fmol/mg cytosol protein. Four more cases of the same variant were later prospectively diagnosed and their high estrogen receptor concentrations correctly predicted. All 12 tumors occurred in postmenopausal women. Most (92%) presented in Stage I or II of the disease. None of the patients died of the tumors during the short period of follow-up (median two years). It is concluded that the alveolar variant of lobular carcinoma of the breast is a tumor with consistently high estrogen receptor concentration and probably a low-grade malignancy. It is also suggested that the tumor may be the lobular equivalent of the ductal infiltrating Comedo carcinoma.

Demonstration of oestrogen receptors in paraffin wax sections of breast carcinoma using the monoclonal antibody 1D5 and microwave oven processing.

This study aimed at assessing the usefulness of a new monoclonal antibody (1D5) for the demonstration of oestrogen receptors (ER) in paraffin wax sections, using brief microwave processing rather than proteolytic predigestion. Routinely processed paraffin wax sections of 50 cases of breast carcinoma with known ER concentrations, estimated by the standard dextran-coated charcoal (DCC) biochemical assay, were examined using the avidin-biotin complex-immunoperoxidase technique. The results were assessed semiquantitatively, using a five grade scoring system. Of the 50 cases examined, 37 were positive and six were negative by both DCC and immunohistology. Of the remaining seven cases, three (6%) were negative by DCC but positive with immunohistology, and four (8%) were positive with DCC and negative with immunohistology. The DCC results of the latter four cases were 10, 14, 14 and 16 fmol/mg protein which is at the lowest level of positivity, our cutoff point being less than 10 fmol. The monoclonal antibody 1D5, as used in this study, can provide easily assessed reliable information about the ER status of breast carcinoma using routinely processed paraffin wax sections.

Nitro blue tetrazolium test in children with kwashiorkor with a comment on the use of latex particles in the test.

The nitro blue tetrazolium test was applied on neutrophils from children with kwashiorkor and from control children. The percentage of formazan cells formed in the kwashiorkor patients was significantly less than in the control group in spite of the presence of associated infections in the former. A direct correlation exists between the percentage of formazan cell formation and total serum proteins, albumin, and haemoglobin. These findings may reflect a decreased bactericidal activity of neutrophils in kwashiorkor. The use of latex particles is an unnecessary technical refinement of the test.

Granular cell myoblastoma: positive staining for carcinoembryonic antigen.

An immunoperoxidase technique for the detection of carcinoembryonic antigen was applied to 10 cases of granular cell myoblastoma. Consistent, strong, intracytoplasmic granular staining, which can be easily interpreted, was obtained in all cases. Schwannomas, neurofibromas, dermatofibromas, and leiomyomas were negative. The test is helpful in confirming doubtful cases. The results tend to support the suggestion that granular cell myoblastoma is derived from perineural rather than endoneural cells.

Immunohistochemical study of lichen planus.

The distribution of IgM and IgG in 20 cases of lichen planus and five cases of non-specific inflammation were studied by the unlabelled antibody-enzyme (PAP) technique. Routine paraffin sections were used. In lichen planus deposits of immunoglobulins were seen in and around epithelial cells, in colloid bodies, at epidermo-dermal junctions, and in some inflammatory cells. All cases examined for IgM and eight out of 13 cases examined for IgG were positive. The peripheral migrating epidermal cells were mostly negative. The application of the PAP technique to skin biopsy and the significance of the findings in lichen planus are discussed.