Design and performance of a double-solenoid magnetic bottle photoelectron spectrometer for attosecond metrology.

The design and performance of an in-house developed double-solenoid magnetic bottle (MB) time-of-flight photoelectron spectrograph are presented. A combination of a strong permanent magnet (Sm2Co17) with a soft iron cone and a double-solenoid geometry is used to generate MB configuration. The first solenoid (length ∼150 mm) is placed inside the vacuum, and the second solenoid (length ∼1 m) is placed outside the vacuum. The double-solenoid geometry improves the effective conductance and reduces overall material outgassing. Due to this, an ultra-high vacuum (∼5 × 10-8 mbar) desirable for the working of the spectrograph was achieved using a small capacity (300 lps) turbo-molecular pump. An optimization of solenoid current generates a smooth magnetic field variation in MB, which keeps the adiabaticity parameter ∼0.6 at ∼25 eV photoelectron energy. The double-solenoid geometry also provides high collection efficiency as well as high energy resolution of the spectrograph. The experimentally measured energy resolution (ΔE) of the spectrograph is better than ∼60 meV at ∼15 eV photoelectron energy. The collection efficiency is estimated to be ∼25% under optimum conditions as compared with ∼10-4 in field-free configuration. The calibrated MB spectrograph is used for the characterization of the attosecond pulse train using a cross-correlation "RABBITT" technique. The attosecond pulse train is generated from 15th to 25th odd high-harmonic orders, in argon filled cell. Attosecond pulses of average duration ∼260 as (FWHM) have been measured. The proposed MB electron spectrograph design provides a compact experimental setup for attosecond metrology and pump-probe studies with a relaxed requirement on vacuum pump capacity.

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.

Randomized trial of a specialist genetic assessment service for familial breast cancer.

BACKGROUND: Because of the growing demand for genetic assessment, there is an urgent need for information about what services are appropriate for women with a family history of breast cancer. Our purpose was to compare the psychologic impact and costs of a multidisciplinary genetic and surgical assessment service with those of current service provisions. METHODS: We carried out a prospective randomized trial of surgical consultation with (the trial group) and without (the control group) genetic assessment in 1000 women with a family history of breast cancer. All P: values are from two-sided tests. RESULTS: Although statistically significantly greater improvement in knowledge about breast cancer was found in the trial group (P: =.05), differences between groups in other psychologic outcomes were not statistically significant. Women in both groups experienced statistically significant reductions in anxiety and found attending the clinics to be highly satisfying. An initial specialist genetic assessment cost pound 14.27 (U.S. $22.55) more than a consultation with a breast surgeon. Counseling and genetic testing of affected relatives, plus subsequent testing of family members of affected relatives identified as mutation carriers, raised the total extra direct and indirect costs per woman in the trial group to pound 60.98 (U.S. $96.35) over costs for the control subjects. CONCLUSIONS: There may be little benefit in providing specialist genetics services to all women with a family history of breast cancer. Further investigation of factors that may mediate the impact of genetic assessment is in progress and may reveal subgroups of women who would benefit from specialist genetics services.

Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival.

Osteopontin (OPN) is a secreted, integrin-binding phosphoprotein that has been implicated in both normal and pathological processes; qualitative increases in OPN blood levels have been reported in a small number of patients with metastatic tumors of various kinds. We measured plasma OPN levels in 70 women with known metastatic breast carcinoma, 44 patient controls who were on follow-up after completion of adjuvant treatment for early breast cancer, and 35 normal volunteers. The median plasma OPN of patients with metastatic disease was 142 microgram/liter (range, 38-1312 microgram/liter) and was significantly different (P < 0.0001, Mann Whitney U test) from both control groups (medians, 60 and 47 microgram/liter; ranges, 15-117 and 22-122 microgram/liter). Furthermore, we found that increasing plasma OPN is associated with shorter survival (P < 0.001) when patients were grouped in terciles for plasma OPN. This was also demonstrated when using a Cox proportional hazards model. Median plasma OPN levels were significantly increased for three or more sites of involvement (median, 232 microgram/liter; n = 13) versus 1 or 2 metastatic sites (medians, 129 and 130 microgram/liter; n = 29 and 28, respectively). Plasma OPN levels were correlated with other biochemical markers related to the extent of disease, such as serum alkaline phosphatase, aspartate succinate aminotransaminase, and albumin (r = 0.81, 0.62, and -0.56, respectively; all P < 0.001). This study demonstrates a statistically significant elevation in plasma OPN in the majority ( approximately 70%) of a large series of patients with metastatic breast cancer when compared (95th percentile) to healthy women or patients who had completed adjuvant treatment for early-stage breast cancer. Furthermore, this is the first study to demonstrate that higher OPN levels in patients with metastatic breast cancer may be associated with an increased number of involved sites and decreased survival.

Prevention of mammary preneoplastic transformation by naturally-occurring tumor inhibitors.

Aberrant hyperproliferation (AH) is a late occurring post-initiational event that precedes mammary tumorigenesis in vivo. Experiments on the spontaneously immortalized, non-tumorigenic murine mammary epithelial C57/MG and MMEC cells were designed to validate AH as an in vitro cellular marker for preneoplastic transformation. Colony forming efficiency (% CFE) in anchorage-independent conditions of growth represented the quantitative parameter for AH. C57/MG and MMEC cells, upon treatment with chemical carcinogens or transfection with oncogenes, exhibited at least a 60-300-fold increase in AH relative to that seen in appropriate untreated controls. Transplantation of mammary epithelial cells initiated either by chemical carcinogens or by oncogenes into mammary fat pads of syngeneic mice produced rapidly growing tumors at the transplant site within 4-6 weeks. The tumor-derived T1/Pr1 and myc3/Pr1 cell lines (positive controls) exhibited at least an 800-900-fold increase in AH. Treatment of initiated cells with naturally occurring tumor inhibitors eicosapentaenoic acid (EPA), indole-3-carbinol (I3C), (-)epigallocatechin gallate (EGCG), squalene (SQE), and perillyl alcohol (PA) at non-toxic doses, resulted in a 70-99% inhibition of AH, depending on the initiator and the chemopreventive test compound. Upregulation of AH in initiated mammary epithelial cells in vitro prior to tumorigenesis in vivo, and persistent inhibition of AH by diverse naturally occurring tumor inhibitors, provides evidence for AH as a cellular surrogate endpoint for induction and modulation of mammary neoplastic transformation.

Quantification of osteopontin in human plasma with an ELISA: basal levels in pre- and postmenopausal women.

OBJECTIVES: To develop an immunoassay for osteopontin (OPN), a secreted phosphoglycoprotein that is implicated in a number of human diseases, and establish basal plasma OPN levels in healthy women. DESIGN AND METHODS: An antigen-capture ELISA was developed to quantity OPN in plasma using a combination of mouse monoclonal and rabbit polyclonal antibodies. Basal OPN levels were determined in blood plasma of 21 pre- and 14 postmenopausal women obtained at 7-day intervals over a 4-week period. RESULTS: A group of 35 healthy women had a median OPN level of 31 micrograms/L (range = 14-64 micrograms/L). Comparison between pre- and postmenopausal women showed that their 4-week average OPN levels did not differ significantly (p > 0.16, Mann-Whitney test), and that levels in each premenopausal individual remained constant during the menstrual cycle, unaffected by cyclical levels of leuteinizing hormone and progesterone. CONCLUSION: Systematic quantification of plasma OPN can now be done by ELISA, which was used to establish basal plasma OPN levels in a group of healthy women. Levels in pre- and postmenopausal women appeared relatively stable over a 4-week period.

Evaluation of general practitioner referrals to a specialist breast clinic according to the UK national guidelines.

The recently published national guidelines to general practitioners for the referral of patients with breast problems were retrospectively applied to letters of all patients attending the Rapid Access Breast Clinic at the University Hospital of Wales. The patients have all had diagnostic investigations performed at the initial visit with a multidisciplinary review of results and provision of a management plan prior to the subsequent visit. Since its inception in May 1995 until the end of the year when the guidelines were published, 2332 new patients had been seen. Overall, 29% of patients with benign breast disease would not have been referred if the guidelines had been strictly followed. Of the 147 symptomatic carcinomas diagnosed from general practitioner referrals (6.3% of total referrals), no invasive cancers would have been missed. One patient with incidental detection of ductal carcinoma in situ (DCIS) in the asymptomatic contralateral breast would not have been referred. Referral for pain without a discrete lump constituted 63% of the patients with a benign diagnosis who fell outside the guidelines. The guidelines also include comprehensive algorithms for the initial management of benign breast symptoms that do not require immediate referral by the general practitioners. Our general practitioners were significantly better at referring patients over 50 years old but the proportion of appropriate referrals were not related to the number referred by each practice. The present guidelines adequately cover referral for the diagnosis of malignant breast disease to a specialist, and may reduce the benign workload of breast clinics.

The National Breast Referral Guidelines have cut down inappropriate referrals in the under 50s.

AIMS: When the National Breast Referral Guidelines were applied to our local GPs letters immediately prior to their release in January 1996, it was shown that on the basis of the GPs own conclusions that 29% of symptomatic women could have been managed initially by their own GP without missing any carcinomas. We conducted this study at the Rapid Access Breast Clinic at the University Hospital of Wales to determine if the breast referral practices of local GPs have altered due to the breast referral guidelines. METHODS: We studied 2332 referrals from the inception of the Rapid Access Clinic in May 1995 to the issue of the guidelines, and 2421 referrals from May 1996 to the end of the year. Random samples of 600 patients were drawn from each year and the referral letters were scored as within or outside the guidelines. Family history patients were excluded. RESULTS: There was an 11% fall in referrals outside the guidelines in the under 50s (chi-squared=<0.001) but the 7% fall in the over 50s was not significant. CONCLUSIONS: The Breast Referral Guidelines seem to have been effective in reducing the higher level of inappropriate referrals in younger patients at less risk of carcinoma.

Osteopontin and p53 expression are associated with tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas.

OBJECTIVE: To examine the association between expression of osteopontin (OPN), p53, other molecular markers (Ki-67, c-erb B2, and estrogen receptor protein) and tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas of the same histology. DESIGN: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections. Plasma OPN level was determined by a quantitative antigen capture assay. SETTING: The patient was seen, treated, and followed up for a period of 5 years at the London Regional Cancer Centre, Ontario, Canada. PATIENT: A 60-year-old woman presented with bilateral infiltrating mammary carcinomas of the same histologic type and grade. Bilateral mastectomy and axillary node dissection showed involvement of 3 of 12 right axillary and 0 of 11 left axillary lymph nodes. She later developed a right chest wall recurrence, followed by widespread metastatic disease to the skull, liver, and left femur. RESULTS: The primary tumor of the right breast was OPN- and p53-positive, whereas the tumor of the left breast was negative for both markers. The development of right axillary lymph node metastases, chest wall recurrence, and distant metastases was associated in all instances with an immunohistochemical profile of high level expression of OPN and p53. Plasma assay for OPN at the time of last admission showed a markedly elevated OPN level. CONCLUSIONS: Increased p53 expression was found to be associated with increased tumor aggressiveness. The association of increased OPN expression with increased malignancy in breast cancer is a novel finding and raises the possibility of a role for OPN in tumor progression, as well as the potential for this marker in predicting clinical aggressiveness.

Significance of heart rate response in shock due to aluminium phosphide poisoning.

Mixed heart rate response has been observed in shock due to aluminium phosphide poisoning. Bradycardia observed in 9 of 30 cases studied indicated transient vagotonia and it reversed with atropine. Both tachycardiac bradycardiac responses were associated with significant increase in the mortality.

Plasma renin activity in shock due to aluminium phosphide poisoning.

Plasma renin activity (PRA) was estimated in 30 patients with aluminium phosphide (AIP) poisoning (study group) admitted in shock. Ten patients in shock other than due to AIP poisoning (Group II A) and 20 normal healthy subjects (Group II B) served as controls. The PRA was significantly higher in the study group and group II A as compared to normal healthy subjects (p less than 0.001). Significantly higher PRA was found in the study group as compared to Group II A (p less than 0.001). The initial higher PRA continued to rise further in the study group but it started decreasing in Group II A as the duration of shock advanced. Continuation of shock in AIP poisoning was probably due to slow release of toxic PH3 gas, which was detected by positive silver nitrate paper test. The rise in PRA was directly proportional to the dose of pesticide consumed. There was direct relationship of mortality with increased PRA. Angiotensin converting enzyme inhibitors may have a role in combating shock in AIp poisoning.

Sequential therapy (triple drug-based induction chemotherapy followed by concurrent chemoradiotherapy) in locally advanced inoperable head and neck cancer patients - Single institute experience.

CONTEXT: India has a high incidence of head and neck squamous cell carcinoma (HNSCC) mostly presenting in advanced stage. In the majority of inoperable patients a combination of chemotherapy and radiotherapy (CRT) is considered as the treatment of choice. Adding induction chemotherapy (ICT) before CRT has shown to decrease systemic relapse. Incorporation of taxanes to the cisplatin and 5-FU-based ICT has shown increase in response rates. AIMS: To evaluate the efficacy and toxicity of triple drug-based ICT followed by CCRT in locally advanced, inoperable HNSCC in the Indian context. SETTINGS AND DESIGN: Prospective, non-controlled, observational study, a single-institute experience. MATERIALS AND METHODS: Consecutive, locally advanced inoperable HNSCC patients were put on sequential therapy consisting of docetaxel, 5-FU and cisplatin for three cycles followed by concurrent weekly cisplatin and radiotherapy for responding or stable disease patients. RESULTS: Forty-four patients were enrolled with male,female ratio of 33/44(75%) and 11/44(25%). Hypopharynx 16/44(36.36%) was the most common site followed by oral cavity 12/44(27.27%) and oropharynx 12/44(27.27%); 38/44(86.36%) patients could complete the planned treatment. Seven patients required dose reduction in ICT. As per the RECIST criteria, 16 patients had Complete Response (CR) and 15 had partial response (PR), 10 had stable disease (SD) and three had progressive disease (PD) after ICT. Thirty-eight patients received concomitant chemo radiotherapy (CCRT); 28/44 (66.63%) patients achieved CR, 10/44 (22.72 %) had PR. The main toxicity was mucositis 18/44 (40.90%) secondary to ICT. Grade III and IV hematological toxicity was seen in 16/44(36.36%), of which 6/44 (13.63%) had febrile neutropenia. CONCLUSIONS: Triple drug-based sequential therapy is tolerable in our context. In this trial from a single institute the results are very encouraging.

Intra-operative imprint cytology for assessing the sentinel node in breast cancer: results of its routine use over 8 years.

INTRODUCTION: Intra-operative imprint cytology (IIC) for analysing sentinel lymph node/s (SLN) in breast cancer allows definitive axillary surgery as a one-step procedure. Most reported studies are research oriented. This study reports long-term results of IIC done as routine clinical practice. MATERIALS AND METHODS: Eight hundred ninety-six female, operable breast cancer patients underwent SLN biopsy over an 8-year period (January 1999-December 2006). Data were extracted retrospectively from medical records. SLNs were sent intra-operatively to the laboratory where they were bisected, touch imprinted and stained with Hematoxylin & Eosin. Patients with positive IIC had axillary clearance. Formal histological analyses of SLNs were compared with IIC findings. The impact of routine pre-operative axillary ultrasound (introduced in 2003) on IIC sensitivity and specificity was also assessed. RESULTS: Median age was 61 years (26-89) and median tumour size was 18 mm (2-100). A total of 244/896 patients had SLN metastases on final paraffin histology of which 177 were correctly detected by IIC (67 false negatives). 39/67 false negatives could be attributed to sampling error. The overall sensitivity and specificity of IIC for the identification of SLN metastases was 73% and 100%, respectively. The sensitivity of IIC after introduction of pre-operative axillary ultrasound decreased from 75% to 71%. DISCUSSION: Routine use of IIC for analysis of the SLN in breast cancer allows complete axillary surgery during a single anaesthetic for a majority of patients undergoing SLN biopsy. Almost two thirds of positive axillae were spared a second operation. False negative results are frequent and patients should be warned about the potential need for further axillary surgery.

No effect of timing of biopsy in the menstrual cycle on incidence of bone marrow micrometastasis in patients with breast cancer.

BACKGROUND: The timing of breast cancer excision relative to the menstrual cycle has been debated to be of significant prognostic value. The differences in survival relative to the timing in the menstrual cycle have been attributed to the incidence of micrometastasis. METHODS: All patients underwent bone marrow aspiration after the diagnostic surgical biopsy, immediately before definitive surgery. The timing of the diagnostic surgical biopsy in the menstrual cycle was calculated according to Senie. Monoclonal antibodies to epithelium-specific antigens were used to detect bone marrow micrometastasis (BMM). RESULTS: This study reports on the effect of the phase of the menstrual cycle on incidence of BMM after surgical biopsy. Ninety-two patients with regular cycles underwent bone marrow aspiration an average of 12 days (range 0-32 days) after biopsy. Thirty-nine patients had undergone biopsy during the follicular phase and 53 patients during the luteal phase. BMM were detected in 31% of patients (29 of 92). We observed BMM in 33% of patients (13 of 39) in the follicular phase and in 30% of patients (16 of 53) in the luteal phase. This difference is not significant (p > 0.70). The mean number of cells detected and the presence of clumps of cells is similar in the two groups. CONCLUSIONS: The incidence and characteristics of bone marrow micrometastases are independent of the timing of diagnostic excision biopsy in the menstrual cycle.

Axillary node dissection in patients with breast cancer diagnosed through the Ontario Breast Screening Program: a need for minimally invasive techniques.

OBJECTIVE: To determine the role of axillary node dissection by studying patient and tumour characteristics of invasive breast cancer through the Ontario Breast Screening Program (OBSP). DESIGN: A retrospective evaluation. SETTING: The London, Ont., branch of the OBSP. PATIENTS: Three groups of women seen were studied: 50 women with screen-detected breast cancers, which were palpable and detected by the nurse-examiner, 62 women with occult screen-detected breast cancers and 353 age matched women with invasive breast cancer from the LRCC prospective database, who served as controls. MAIN OUTCOME MEASURE: The proportion of involved axillary nodes within the 3 groups based on patient and tumour characteristics. RESULTS: Twenty-five (22.3%) of the 112 women had screen-detected tumours less than 1 cm in dimension, but only 1 had an involved axillary node. Twelve (19%) of the 62 women with occult screen-detected tumours had involved lymph nodes compared with 17 (34%) of the 50 women with palpable screen-detected cancers (NS). In the control group tumour dimension less than 1 cm versus 1 cm or larger had a marked effect on the probability of axillary node involvement (12.5% v. 40.7%, p = 0.001). In the palpable screen-detected group 3 times as many women with outer quadrant or central lesions had involved nodes as those with inner quadrant lesions (38% v. 12%) and for those with a family history of breast cancer almost twice as many had involved axillary nodes. In occult screen-detected patients there was more nodal involvement in patients aged 60 years or less than in those older than 60 years (35% v. 10%, p = 0.042); only 4 of 41 patients older than 60 years had involved nodes at surgery. A significant difference in nodal involvement was found with respect to high or intermediate grade versus low grade lesions in the occult group (44% v. 12%, p = 0.033). In the control group, tumour grade (intermediate and high [45.3%] v. low [20.0%]) and hormone replacement therapy (HRT) (current or recent use [56.5%] v. no use [34.5%]) were significant findings (p < 0.001 and p = 0.005 respectively). CONCLUSIONS: Women older than 60 years with tumours smaller than 1 cm had a low probability of nodal positivity (0% to 8.7%), but there is insufficient information in this group to give a 95% or better prediction of nodal status at the time of surgery. Studies of minimally invasive techniques such as sentinel node biopsy are needed in this group to minimize surgical morbidity in these women who, as a result of early diagnosis, have an excellent long-term outlook.

Endocrine characteristics of human breast epithelial cells, MCF-10F.

MCF-10F is a spontaneously immortalized nontransformed human breast epithelial cell line which does not grow in soft agar or form tumors in nude mice. Though the presence of estrogen receptors has not been found in these cells, they can metabolize estradiol very efficiently. The present study describes the endocrine characteristics of this cell line with respect to growth response to estradiol and its metabolites, estradiol metabolism and aromatase activity. MCF-10F cells were growth stimulated by 16alpha-hydroxyestrone and estriol, whereas, estradiol and other estradiol metabolites did not affect cell proliferation. The constitutive level of 16alpha-hydroxyestrone, a metabolite of estradiol biotransformation that has been associated with enhanced carcinogenesis in several animal, cell and tissue culture models, was a hundredfold higher in the non-transformed MCF-10F cells than in the transformed MCF-7 cells. Treatment with the carcinogen, dimethylbenz(a)anthracene (DMBA), however, did not upregulate 16alpha-hydroxylation as was observed in transformed MCF-7 cells. MCF-10F cells also had no detectable aromatase activity though the level of 17-oxidation was unusually high as compared with MCF-7 cells. Our results using the non-transformed MCF-10F cells as a model system suggests that the presence of high level of 16alpha-hydroxyestrone, a metabolite previously shown to be associated with malignant phenotype, may not be sufficient for breast cancer transformation.

Osteopontin expression in a group of lymph node negative breast cancer patients.

The aim of this study was to examine the cellular distribution of osteopontin (OPN) protein [by immunohistochemical (IHC) analysis] and mRNA [by in situ hybridization (ISH)] in the primary tumors of lymph node negative (LNN) breast cancer patients and to determine whether the level of immunodetectable OPN may be associated with tumor aggressiveness. We examined OPN levels in tumors from 154 patients with LNN breast cancer who were followed for a median of 7 years (range 1.7-16.3 years). IHC staining for OPN was seen in tumor infiltrating macrophages and lymphocytes in 70% of these tumors, and in the carcinoma cells themselves in 26%. ISH was performed to determine cellular distribution of OPN mRNA expression in sections from selected tumors. OPN mRNA was detected in groups of tumor cells, individual tumor cells and tumor infiltrating macrophages and lymphocytes. Matched sections showed that some tumor cells with IHC staining for OPN protein were also positive for OPN mRNA by ISH, in contrast with previous studies which have shown OPN mRNA expression only in tumor infiltrating inflammatory cells. Our results thus indicate that OPN protein can be produced by breast cancer cells in vivo and suggest that it may also be taken up from the environment (i.e., secreted by inflammatory cells or other tumor cells). Tumor cell IHC staining intensity was then assessed using a semiquantitative scoring system. Univariate analysis showed tumor cell OPN positivity above an optimized cutpoint to be significantly associated with decreased disease-free survival (DFS) and overall survival (OS). The results of this pilot study thus suggest that the ability of breast cancer cells to either synthesize OPN or to bind and sequester OPN from the microenvironment may be associated with tumor aggressiveness and poor prognosis.