Neuropeptide reporter assay for serum, capillary blood and blood cards.

In the associated main paper (JPBA 2019), substance P was shown to be a valuable neuropeptide reporter substance to monitor the protease activity of serum. The assay was developed based on the predecessor assay using bradykinin (JPBA 2017). Both neuropeptides are of interest in inflammation and pain research and were thus explored for use with capillary blood and blood cards. Here, we present the protocols and set them in perspective to above neuropeptide assays for serum.•Neuropeptide reporter substance protease activity assay for use with fresh and dried blood.•Dabsylated Substance P and bradykinin are substrates of angiotensin-converting enzyme and other proteases.•Neuropeptides of interest in inflammation and pain.

Evidence for a white matter lesion size threshold to support the diagnosis of relapsing remitting multiple sclerosis.

BACKGROUND: The number of white matter lesions (WML) in brain MRI is the most established paraclinical tool to support the diagnosis of multiple sclerosis (MS) and to monitor its course. Diagnostic criteria have stipulated a minimum detectable diameter of 3 mm per WML, although this threshold is not evidence-based. We aimed to provide a rationale for a WML size threshold for three-dimensional MRI sequences at 3 T by comparing patients with relapsing-remitting MS (RRMS) to control subjects (CS). METHODS: We analyzed MR images from two cohorts, obtained at scanners from two different vendors, each comprising patients with RRMS and CS. Both cohorts were examined with FLAIR and T1w sequences. In total, 232 patients with RRMS (Expanded Disability Status Scale: mean = 1.6 ± 1.2; age: mean = 36 ± 10) as well as 116 age- and sex-matched CS were studied. We calculated odds ratios across WML volumes. The WML size threshold, which discriminated best between patients and CS, was estimated with receiver operating characteristic curve analysis. RESULTS: In both cohorts, odds ratios increased continuously with increasing WML volumes, and discriminative power was highest at a WML size threshold corresponding to a diameter of about 3 mm. CONCLUSION: The stipulated WML size threshold of 3 mm in diameter for the diagnostic criteria of MS seems a reasonable choice for three-dimensional MRI sequences at 3 T.

The evolution of the phage shock protein response system: interplay between protein function, genomic organization, and system function.

Sensing the environment and responding appropriately to it are key capabilities for the survival of an organism. All extant organisms must have evolved suitable sensors, signaling systems, and response mechanisms allowing them to survive under the conditions they are likely to encounter. Here, we investigate in detail the evolutionary history of one such system: The phage shock protein (Psp) stress response system is an important part of the stress response machinery in many bacteria, including Escherichia coli K12. Here, we use a systematic analysis of the genes that make up and regulate the Psp system in E. coli in order to elucidate the evolutionary history of the system. We compare gene sharing, sequence evolution, and conservation of protein-coding as well as noncoding DNA sequences and link these to comparative analyses of genome/operon organization across 698 bacterial genomes. Finally, we evaluate experimentally the biological advantage/disadvantage of a simplified version of the Psp system under different oxygen-related environments. Our results suggest that the Psp system evolved around a core response mechanism by gradually co-opting genes into the system to provide more nuanced sensory, signaling, and effector functionalities. We find that recruitment of new genes into the response machinery is closely linked to incorporation of these genes into a psp operon as is seen in E. coli, which contains the bulk of genes involved in the response. The organization of this operon allows for surprising levels of additional transcriptional control and flexibility. The results discussed here suggest that the components of such signaling systems will only be evolutionarily conserved if the overall functionality of the system can be maintained.

The impact of preoperative breast biopsy on the risk of sentinel lymph node metastases: analysis of 2502 cases from the Austrian Sentinel Node Biopsy Study Group.

Preoperative breast biopsy might cause disaggregation of tumour cells and tumour cell spread. The purpose of this study was to investigate the impact of preoperative biopsy on the rate of metastases to the sentinel lymph node (SLN) of patients with primary breast cancer. We report the results of 2502 patients with primary breast cancer, who were operated, and a sentinel node biopsy was performed. The association of preoperative biopsy with the risk of SLN metastases was examined by regression analyses and tested for possible confounding well-known factors for axillary node metastases. In all, 1890 patients were available for final analyses; 1048 (55.4%) patients had a preoperative diagnosis performed by fine-needle aspiration or core biopsy; 641 (33.9%) patients had a positive SLN when conventional H&E and IHC staining was performed. Patients with preoperative breast biopsy showed a 1.37 times (95% CI, 1.13-1.66) increased risk of SLN metastases on univariate analysis, but this result was not persistent when analysis was adjusted for other relevant factors for axillary node metastases, OR 1.09 (95% CI, 0.85-1.40). In addition, subgroup analyses of the risk for occult micro metastases to the SLN (detected by IHC only) on H&E-negative cases also showed no increased risk associated with preoperative biopsy, OR 1.07 (95% CI, 0.69-1.65). The conclusion, based on the present data, is that preoperative breast biopsy does not cause artificial tumour cell spread to the SLN, with possible negative impact on the prognosis of breast cancer.

Malignant pleural and pericardial effusion in invasive breast cancer: impact of the site of the primary tumor.

BACKGROUND: Malignant effusion in invasive breast cancer, either pleural or pericardial, is associated with a poor survival rate. We investigated the role of the location of invasive breast cancer in developing malignant pleural or pericardial effusion. METHODS: Three thousand eight hundred and fifty six women with a history of invasive breast cancer between 1960 and 1994 were analyzed in a retrospective study. Two hundred and six patients (5.34%; group A) developed malignant pleural and/or pericardial fluid as the first visible clinical sign of distant metastasis. A control group of 206 patients (group B) without malignant effusions were matched in terms of clinical and pathological characteristics. The two groups were compared in respect of the location of the primary tumor. Fifty patients in group A (n = 50; 24.2%) had tumors in the inner quadrants, 83 patients (n = 83; 40.3%) in the outer quadrants, and 13 patients (n = 13; 6.3%) in a central location. No data were available for 60 patients (n = 60; 29.1%). In group B, 12 patients (n = 12; 5.9%) had tumors in the inner quadrants, 140 patients (n = 140; 68.0%) in the outer quadrants, 14 patients (n = 14; 6.8%) had centrally located tumors, while no data were available for 40 patients (n = 40; 19.4%). Invasive ductal carcinomas (IDC) located in the inner quadrants were highly significantly associated with increased pleural or pericardial effusion as the first site of distant metastasis (p

Clear cell carcinoma arising in endometriosis of the rectum following progestin therapy.

A 40-year-old woman presented at our hospital with rectal stenosis. Computed tomography demonstrated a conglomerate tumor in the pelvis and malignant look-alike lesions in the liver and in both lungs. A palliative deep rectum resection was done. Histologically, clear cell carcinoma with a small area of endometrioid carcinoma was diagnosed. Severe endometriosis had been diagnosed 13 years earlier. The patient had been treated with medroxyprogesterone acetate (MPA) for two years; no estrogen therapy had been given. The association between unopposed estrogen replacement therapy and malignant transformation of endometriosis is documented, but malignant transformation following progestin therapy has not been reported previously. It appears that radical surgery is the only means of preventing malignant transformation of the lesion in patients with endometriosis.

Lunar phases and survival of breast cancer patients--a statistical analysis of 3,757 cases.

The potential influence of lunar phases on human life has been widely discussed by the lay press. The purpose of this study was to find out whether the timing of surgery during particular lunar phases influences the survival of breast cancer patients. It has been postulated that breast cancer surgery performed during the waxing moon, or particularly at full moon, is associated with a poorer outcome. We tested this hypothesis by evaluating the overall survival for 3,757 consecutive patients with invasive breast cancer. All patients underwent either modified radical mastectomy or breast conserving surgery plus radiotherapy, followed by adjuvant cytotoxic or hormonal therapy. The date of definitive surgery was allocated to the lunar phases. 1,904 (50.7%) patients were operated on during the waxing moon and 1,853 (47.3%) during the waning moon. The median follow-up was 74 months (range 1-372 months). The mean age at primary surgery did not differ significantly in the two groups 58.39 (SD 13.14) versus 58.34 (12.75) (p >0.05, t-test). Breast cancer stages at initial diagnosis were evenly distributed according to the lunar phases (p = 0.325; chi-square). Survival curves were plotted according to the method of Kaplan-Meier. No significant differences were observed when timing of surgery was allocated to the lunar phases (p = 0.4841, log-rank). Subgroup analysis of premenopausal patients revealed similar results (p = 0.2950, log-rank; n = 1072). Using multivariate Cox modelling, we found a significant association between the patient's age, stage of disease and survival, whereas no association with survival was observed for the timing of surgery (RR= 1.062; 95% CI, 0.970-1.163; p = 0.1937). No significant differences in overall survival of breast cancer patients were observed when timing of breast cancer surgery during the lunar cycle was considered. Although this was not a prospective randomized trial, the statistical magnitude of the results do not support any recommendations for scheduling patients for surgery at any particular day of the lunar phase.

Does long-term application of granulocyte colony-stimulating factor adversely influence overall survival in patients with ovarian cancer? A clinical study.

The purpose of this study was to investigate the effect of long-term administration of G-CSF with regard to its impact on overall survival of patients with ovarian cancer. We report the results of a non-randomized trial on 64 patients with advanced ovarian cancer treated with 6 cycles of conventional chemotherapy. Chemotherapy comprised carboplatin 400 mg/m2 and epirubicin 70 mg/m2 on day 1 of each cycle and prednimustine 100 mg/m2 on days 3 to 7, every 28 days. Thirty-three patients received CEP chemotherapy with G-CSF support whereas 31 women received CEP chemotherapy alone. The schedule of G-CSF was 5 mg/kg/day subcutanously on days 8 to 21 of each cycle. The severity of reduction in white cells and neutrophil count was significantly different in the two treatment groups (p<0.05), with more toxicity in the non- G-CSF group. G-CSF users had a non significant 0.88-fold lower risk of dying from ovarian cancer (95% CI, 0.48-1.60, p=0.678). In a survival analysis using a Cox proportional hazards model, residual tumor remained as an independent prognostic factor. The increasing amount of residual tumor resulted in a 1.767-fold higher risk (95% CI, 1.23-2.53, p=0.002) of death secondary to the underlying disease. In conclusion, this trial has failed to demonstrate any negative impact on patients' overall survival for the additional use of G-CSF with platinum-based chemotherapy; our results were consistent with the beneficial effects of G-CSF treatment on cytotoxic chemotherapy-induced myelosuppression.

First report of lymphatic mapping with isosulfan blue dye and sentinel node biopsy in cervical cancer.

BACKGROUND: Sentinel lymph node status provides important information about the status of the regional nodes in various malignant tumors. Our report describes a method of identifying the sentinel lymph nodes in cervical cancer. PATIENTS AND METHODS: In three cases of early cervical cancer, isosulfan blue dye was injected paracervically into each lateral fornix immediately before surgery. RESULTS: In all cases we identified two to three blue stained (sentinel) lymph nodes located either at the iliac artery or in the obturatory space. The blue colored nodes were positive for disease, all other pelvic lymph nodes removed were negative. CONCLUSIONS: Our findings demonstrate that preoperative lymphatic mapping with vital blue dye is an easy to perform technique to visualize sentinel lymph nodes in cervical cancer. Sentinel lymph node status may be representative of the pelvic lymph node status in cervical cancer and thus could provide important information for further treatment.

Significance of pretreatment serum hemoglobin and survival in epithelial ovarian cancer.

Tumor anemia is common in patients with malignant tumors and it was repeatedly demonstrated to be associated with impaired prognosis in patients with malignant tumors. We conducted a retrospective analysis based on 553 patients with histologically proven epithelial ovarian cancer. Blood hemoglobin levels were determined before surgery and patients with values <12 g/dl were considered anemic. Data analysis included univariate and multiple Cox models. Tumor anemia was present in 143 (25.9%) patients before surgery. Tumor anemia was present in 143 (25.9%) patients before surgery. In a multivariate Cox model, pretreatment hemoglobin values proved to be an independent prognostic factor for patients with stage I-II epithelial ovarian cancer (n=203), but failed to attain significance in patients with stage III-IV disease (n=350). Tumor anemia defined as pretreatment hemoglobin values <12 g/dl may indicate patients with stage I and II epithelial ovarian cancer, who are at increased risk of relapse.

CA 125 regression after two completed cycles of chemotherapy: lack of prediction for long-term survival in patients with advanced ovarian cancer.

The prognostic influence of CA 125 regression between the time point before surgery and after two completed courses of chemotherapy was studied in 210 patients with advanced ovarian cancer, and was compared to other well established prognostic factors. CA 125 blood samples were collected preoperatively (CA 125 pre) and 3 months after surgery (CA 125 3 mo) (at the beginning of the 3rd cycle of chemotherapy). The parameter CA 125 regression defined as log10 (CA 125 3 mo/CA 125 pre) was used for statistical analysis. In a survival analysis using a Cox proportional hazards model, CA 125 regression (P = 0.0001), residual tumour (P = 0.0001), age (P = 0.0095) and grading (P = 0.044) were independent variables, whereas stage of disease, histology, ascites and type of surgery failed to retain significance. Using log10 (CA 125 3 mo/CA 125 pre) as simple covariate in a Cox model showed a hazard ratio of 1.70 (95% confidence interval 1.32-2.19, P = 0.0001). However, a detailed analysis of the interaction of time with the prognostic factor CA 125 regression on survival revealed a strong time-dependent effect with a hazard ratio of more than 6 immediately after two courses of chemotherapy, whereas within approximately 1 year the hazard ratio for the surviving patients dropped quickly to the neutral level of 1. In summary, CA 125 regression is an independent prognostic factor for survival of women with advanced ovarian cancer and allows an identification of a high-risk population among patients with advanced ovarian cancer. However, the discriminating power of serial CA 125 for long-term survival seems to be temporary and prediction of individual patients outcome is far less precise.

Association between endometrial cancer and tamoxifen treatment of breast cancer.

A retrospective cohort-study in 4109 breast cancer patients was undertaken to determine how tamoxifen affected the risk of endometrial cancer. Data on 1701 tamoxifen-treated women were analysed. Two thousand four hundred and eight non-tamoxifen users served as control group. The occurrence of new primary uterine cancers was assessed by computerized linkage to the Austrian Cancer Registry. Twenty-five women who subsequently developed endometrial cancer were identified. Eight uterine cancers occurred in the tamoxifen group, whereas 17 uterine cancers were found in the control group. The estimate of the relative risk (RR) showed an increased risk to develop endometrial cancer for the tamoxifen group RR 1.136 (95% CI 0.71; 1.80). Analysis of relevant confounding variables did not show any differences in the two groups. In conclusion, this retrospective study demonstrated a non-significant increased risk of endometrial cancer in women receiving tamoxifen as treatment for breast cancer. However, the magnitude of RR and the absolute number of endometrial cancer cases in this long term observation demonstrate clearly that the clinical benefit of tamoxifen therapy greatly outweighs the risk.

Tumor flow in malignant breast tumors measured by Doppler ultrasound: an independent predictor of survival.

The purpose of this study was to investigate tumor blood flow in breast cancers with regard to its impact on the overall survival of patients. Tumor blood flow was assessed in seventy-four patients with primary breast cancer by the use of color-coded Doppler ultrasound techniques. Preoperatively obtained Doppler frequency spectra were analyzed for peak systolic flow velocity (Vmax). Color Doppler signals were detected in 71 (96%) of the breast tumors. Out of 74 patients, 17 experienced a relapse or distant metastasis, and 15 women had died due to breast cancer at the time of data analysis. The mean Vmax of the patients who had died was 0.27 m s(-1), whereas survivors showed a mean Vmax of 0.16 m s(-1) (p = 0.01). Vmax, nodal status, and progesterone receptor status remained the only significant factors of overall survival in the multivariate model, whereas tumor size, tumor grade, and estrogen receptor status failed to retain prognostic significance. Moreover, Vmax was identified as the most important prognostic marker for survival in our series. The five-year-survival was 82.3% in Vmax < or = 0.25 m s(-1) patients versus 36.6% in women with tumor flow greater than 0.25 m s(-1). Patients with Vmax > 0.25 m s(-1) experienced a 4.33-fold increased risk of death secondary to the underlying disease. In summary, our data showed that tumor blood flow velocity measured by ultrasonography is an independent prognostic factor of survival in breast cancer patients. Furthermore, tumor flow velocity allows identification of patients at very high risk of death due to breast cancer. Large scale clinical trials should evaluate the clinical usefulness and future impact of this procedure for adjuvant treatment decisions.

Color-coded and spectral Doppler flow in breast carcinomas--relationship with the tumor microvasculature.

The phenomenon of tumor angiogenesis is an important aspect of understanding tumor biology. Studies in breast carcinoma have shown microvessel density (MVD) assessed by immunohistochemistry to be of prognostic importance in primary breast cancer. On the other hand, recently developed highly sensitive color-coded Doppler techniques offer a noninvasive method to examine neovascularisation in breast tumors. The purpose of this study was to determine the relationship between Doppler flow parameters and microvessel count assessed by immunohistochemistry. Fifty-three patients with primary breast cancer were examined preoperatively with color-coded Doppler ultrasound. The obtained Doppler frequency spectra were analyzed for peak systolic flow velocity (Vmax). Following surgery, paraffin-embedded microsections were immunohistochemically stained for factor VIII-related antigen. Tumor angiogenesis was assessed by microvessel count under light microscopy. Undifferentiated tumors correlated with high MVD (p=0.009) whereas other clinicopathological parameters were not associated with MVD. Color Doppler signals were detected in 50 out of 53 breast tumors. Evaluation of tumor flow velocity with various clinicopathological parameters showed a significant correlation with tumor size (p=0.0001) and lymph node metastasis (p=0.02). However, there was no significant correlation between MVD and intratumoral blood flow velocity assessed by color-coded Doppler. Our findings showed that Doppler flow measurement did not correlate with the extent of tumor angiogenesis of breast cancer. The present data give circumstantial evidence that microvessel count assessed by immunohistochemistry reflects the microvascular network, whereas tumor vasculature documented by Doppler ultrasound supplies information on the macrovasculature.

TATI (tumor associated trypsin inhibitor) and cancer antigen 125 (CA 125) in patients with early-stage endometrial cancer.

BACKGROUND: In patients with gynecologic malignancies, a 6 kD polypeptide known as the tumor-associated trypsin inhibitor (TATI) is present in high concentrations, both in the urine and the serum. This study attempts to evaluate the usefulness of pretreatment serum levels of TATI (cutoff level 21 ng ml-1) and CA 125 (cutoff levels 35 U ml-1 and 65 U ml-1) in the prediction of early endometrial cancer. PATIENTS AND METHODS: One hundred twenty-seven patients with stage I and II endometrial carcinomas, 110 healthy women and 258 women with benign pelvic pathologies were evaluated. The data obtained were correlated with the tumor stage and tumor grade. RESULTS: Overall, TATI showed a sensitivity of 31% and a specificity of 81%. The sensitivity and specificity of CA 125 > 35 U ml-1 was 25% and 86%, respectively. When both serum tumor markers were combined the sensitivity increased to 48% (CA 125 > 35 U ml-1), with a specificity of 71%. A correlation with the depth of myometrial infiltration was found for neither of the tumor markers under investigation. In addition, neither TATI nor CA 125 correlated well with tumor grade. The combination of TATI and CA 125 had a high positive predictive value (84%) when no other gynecologic pathologies were present. Furthermore, if TATI and CA 125 levels are within normal ranges and gynecological examination does not show other abnormalities besides vaginal bleeding, endometrial carcinoma appears to be very unlikely. CONCLUSION: We concluded that, while TATI and CA 125 may not be recommended as a screening method for the detection of endometrial cancer, the combination of TATI and CA 125 is a valuable additional tool for further evaluation of women with suspected uterine cancer.

Serum levels of the tumour-associated trypsin inhibitor in patients with endometriosis.

OBJECTIVE: To show that in patients with endometriosis a 6 kD polypeptide, the tumour-associated trypsin inhibitor (TATI), can occur at elevated concentrations in serum. DESIGN: In a prospective study TATI serum levels were assessed prior to surgery in 368 consecutive patients suffering from benign gynaecological diseases (e.g. pelvic pain, infertility, elective sterilisation, uterine fibroids and pelvic masses) with (n = 71) and without (n = 297) endometriosis, who underwent laparoscopy or laparotomy for diagnosis and/or treatment. RESULTS: The TATI serum levels of patients suffering from endometriosis were significantly different from those in the control group and showed a positive correlation with the stage of endometriosis. The sensitivity of TATI was 0.34 with a specificity of 0.85 for all cases of endometriosis, with an increase of sensitivity (0.67) and slight decrease of specificity (0.82), considering only a group of Stage III/IV patients. Excluding patients with benign ovarian cysts, the specificity of TATI was 0.91 and 0.85, respectively. The combination of TATI and CA125 showed an increase of sensitivity to 0.59 for all cases of endometriosis and 0.89 for patients with Stage III/IV endometriosis. CONCLUSIONS: The sensitivity of TATI as a screening method for endometriosis is too low, but considering its high specificity, TATI in combination with CA125 could provide an additional diagnostic tool in diagnosis and follow up of patients with endometriosis.

Endometrial cancer after tamoxifen treatment: a descriptive study of 25 breast cancer patients who subsequently developed endometrial cancer.

The aim of this study was to determine whether tamoxifen affects the histopathological features and clinical outcome of endometrial cancer occurring after breast cancer. 25 patients with a history of breast cancer who subsequently developed endometrial cancer were identified by computer assisted linkage of the Lainz Medical Center Tumor Registry with the National Austrian Cancer Registry. 8 endometrial cancers were tamoxifen-associated, whereas 17 cases were not. The median time of tamoxifen use was 32 months, with a median cumulative tamoxifen dose of 27 g. Detailed histopathological data did not show substantial difference between the two groups. The distribution by stage (FIGO) in the tamoxifen group was Stage I, 7 (87.5) with 1 patient unstaged (12.5%); and Stage I, 13 (76%), II, 2 (12%) and, stage III 2 (12%) for the non-tamoxifen users. No risk of high-grade endometrial lesions with a poor prognosis was found. The only suggestion of a difference was a trend for the mean interval between detection of breast and endometrial cancer to be shorter in the tamoxifen group (50.5 vs. 88.1 months p = 0.07). The conclusion to be drawn from this study appears to be that endometrial cancers occurring after tamoxifen exposure are of the same tumor type. Due to the small number of cases described we were not able to disprove the hypothesis that tamoxifen associated tumors were different in terms of clinical outcome and survival. Regular gynecological examinations are recommended for all breast cancer patients.

The application of fibrin glue after axillary lymphadenectomy in the surgical treatment of human breast cancer.

Experimental studies point out that a reduction of lymph flow can be obtained by the local application of fibrin glue following axillary lymphadenectomy in the surgical treatment of breast cancer. In a prospective study the influence of human fibrin glue on postoperative axillary lymph secretion and the period of drainage of the wound cavity were evaluated. In 40 patients, 5 ml of fibrin glue (Tissucol) was applied to the wound cavity by the use of a spray applicator (Tissumat) immediately after axillary dissection of the lymph nodes. For drainage of the wound area Redon suction-drains were used. The daily amount of postoperative lymph secretion was measured and drains were removed at a lymph secretion of less than 20 ml. 40 patients who underwent surgery and axillary lymphadenectomy without subsequent application of fibrin glue sourced as control group. No significant difference concerning the total amount of lymph secretion, the mean period of drainage or the incidence of lymphatic cysts was observed. In our study, the expected occlusion of the wound cavity by the application of fibrin glue after axillary lymphadenectomy did not lead to any advantage when compared with the control group.

Tumor-associated trypsin inhibitor (TATI) and cancer antigen 125 (CA125) in patients with epithelial ovarian cancer.

In 180 patients with epithelial ovarian cancer and 214 women with benign pelvic pathologies, serum levels of TATI (cut-off point 21 ng ml-1) and CA 125 (cut-off point 35 U ml-1) were determined. Data were correlated with tumour stage, histological type and tumour grade. Overall, when used as a single marker, TATI showed a sensitivity of 63% and a specificity of 72%, whereas the sensitivity and specificity of CA 125 > 35 U ml-1 were 80% and 82% respectively. A combination of the two markers increased the sensitivity to 91% (TATI > 21 ng ml-1 or CA 125 > 35 U ml-1), whereas the specificity decreased to 65%. TATI was clearly superior in diagnosing mucinous carinomata of the ovaries; the rate of true positive findings was 64% versus 50% for CA 125. Unlike CA 125, TATI levels correlated well with tumour grade. In conclusion, CA 125 remains the single tumour marker of choice in the diagnosis of malignant epithelial ovarian cancer, while TATI appears to be a valuable complementory marker with a higher sensitivity in cases of poorly differentiated and mucinous carcinomata.