Tartrate-resistant acid phosphatase 5b as a serum marker of bone metastasis in breast cancer patients

Diagnosis and follow-up of bone metastases in breast cancer patients usually rely on symptoms and imaging studies. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is a specific marker of osteoclasts and is herein proposed as a marker of bone metastasis in breast cancer patients. An immunoassay using a monoclonal antibody, 14G6, was used to measure the activity of serum TRACP 5b at pH 6.1 in 30 early breast cancer patients without bone metastasis and in 30 aged-matched breast cancer patients with bone metastasis. Another 60 normal volunteers were recruited as controls. Bone alkaline phosphatase (BAP), a traditional marker of bone turnover, was also measured in selected cases. The overall mean TRACP 5b activity in normal women was 2.83 ± 1.1 U/I, and it increased with age. The mean TRACP 5b activity in early breast cancer patients did not differ from that of the normal group (2.93 ± 0.64 vs. 2.83 ± 1.1 U/I; p=0.66), whereas it was significantly higher in breast cancer patients with bone metastasis (5.42 ± 2.5 vs. 2.83 ± 1.1 U/I; p<0.0001). BAP activity was significantly higher in breast cancer patients with bone metastasis than in early breast cancer patients (p=0.004). Serum TRACP 5b activity correlated well with BAP activity in breast cancer patients with bone metastasis (p<0.0001), but not in normal individuals or in patients without bone metastasis. TRACP 5b activity can be considered a surrogate indicator of bone metastasis in breast cancer patients.     

Patterns of initial recurrence and prognosis after sentinel lymph node biopsy and selective lymphadenectomy for melanoma

The histologic status of the sentinel lymph node is a highly significant prognostic factor for patients with clinically localized cutaneous melanoma. The patterns of initial treatment failure of patients with positive sentinel lymph node biopsy versus those with negative results have not been well described. The purpose of this study was to determine the relative prognostic importance of sentinel lymph node status and to compare patterns of initial treatment failure and prognosis of node-positive versus node-negative cutaneous melanoma patients staged by sentinel lymph node biopsy and selective lymphadenectomy. The authors reviewed the pertinent demographic and surgical data in a consecutive series of patients with cutaneous melanoma who underwent sentinel lymph node staging of nonpalpable regional nodes. Sentinel lymph node biopsy was performed using a combination of blue dye and radiolocalization. Patients with positive biopsy results underwent selective lymphadenectomy, whereas those with negative results were observed. Site(s) and date(s) of initial recurrence and death were determined, and disease-free and overall survival probabilities were compared between positive and negative groups using the log-rank test and multivariable Cox regression analysis. Between February of 1994 and August of 2000, 408 patients with melanoma underwent sentinel lymph node biopsy to stage 518 regional lymph node basins. Mean Breslow tumor thickness was 2.27 mm (range, 0.2 to 14.0 mm). Eighty-five patients (20.8 percent) had at least one histologically positive sentinel lymph node, and selective lymphadenectomy yielded additional positive lymph nodes in 18 of 84 patients (21.4 percent). Recurrences were noted in 70 patients (17 percent) at a median follow-up period of 31.4 months. Recurrences were more frequent in patients with positive biopsy results (36.5 percent) than in those with negative results (12.1 percent, p < 0.0001). Distant sites of initial recurrence were more likely in the positive group than in the negative group (71 percent versus 49 percent of recurrences, respectively; p = 0.06). The false-negative rate for sentinel lymph node staging was 4.5 percent and overall accuracy was 99 percent compared with clinical follow-up. Disease-free and overall survival correlated significantly with tumor thickness, ulceration, sentinel lymph node status, and the number of tumor-positive lymph nodes (two-sided p < 0.0001 for all comparisons). Multivariable analysis revealed that sentinel lymph node status (p = 0.003), tumor thickness (p = 0.016), ulceration (p = 0.006), and age (p = 0.003) were significant independent predictors of survival for the entire group. Tumor thickness and ulceration were significant predictors of recurrence and survival in sentinel node-negative patients but not in sentinel node-positive patients. Sentinel lymph node histology is possibly the most important negative predictor of early recurrence and survival in patients with American Joint Committee on Cancer stage I and II melanoma. The number of positive lymph nodes provides additional prognostic information. Although sentinel node-negative patients are a prognostically favorable group, various combinations of local and regional recurrences comprise the most common pattern of initial relapse after a negative sentinel lymph node biopsy result.     

Significant effects of biologic therapy on lipid profiles and insulin resistance in patients with rheumatoid arthritis

IntroductionThe goal of this study was to investigate (1) the associations of rheumatoid arthritis (RA)-related inflammation or rheumatoid factor/anti-cyclic citrullinated peptide (anti-CCP) positivity with lipid profiles and insulin resistance (IR), (2) the effects of biologic therapy on lipid profiles and IR, and (3) potential predictors for the presence of subclinical atherosclerosis.MethodsSerum levels of lipid profiles were determined by enzymatic methods in 32 adalimumab-treated patients, 16 etanercept-treated patients, 24 tocilizumab-treated patients, and 20 biologic-naïve patients. Atherogenic index, which corresponds to the ratio of total cholesterol to high-density lipoprotein cholesterol (HDL-C), was calculated. IR was measured by homeostasis model assessment. Pro-inflammatory cytokine levels were examined by enzyme-linked immunosorbent assay. Common carotid artery intima-media thickness was determined by using sonography.ResultsThere was an inverse correlation between disease activity (disease activity score for 28 joints, or DAS28) and low-density lipoprotein cholesterol (LDL-C) levels (r = −0.226, P <0.05) and a positive correlation between DAS28 and IR (r = 0.361, P <0.005). Anti-CCP-positive patients had significantly higher DAS28 and IR compared with anti-CCP-negative patients. There was also a positive correlation between IR and levels of interleukin-6 or tumor necrosis factor-alpha (TNF-α). HDL-C levels significantly increased in patients receiving 6-month anti-TNF-α therapy, and levels of total cholesterol, LDL-C, and triglyceride increased in tocilizumab-treated patients. IR significantly decreased in patients under biologic therapy but was unchanged in biologic-naïve patients. Age, IR, and DAS28 were significant predictors of severe subclinical atherosclerosis (odds ratios of 1.08, 2.77, and 2.52, respectively).ConclusionsSignificant associations of RA-related inflammation with lipid profiles and IR indicate the involvement of RA in atherosclerosis pathogenesis. Biologic therapies were associated with IR reduction without change in atherogenic index, but their beneficial effects on atherosclerosis reduction need to be verified in the future.     

Expression of matrix metalloproteinase-9 (gelatinase B) in gouty arthritis and stimulation of MMP-9 by urate crystals in macrophages

To investigate the relevance of gelatinase-B (matrix metalloproteinase 9, MMP-9) in gouty arthritis (GA), we tested the occurrence of MMP-9 in GA patients and cell culture system. Gelatinolytic activity in the synovial fluid (SF) of patients with different kinds of arthritis was assessed by gelatin zymography. A predominant 92-kDa MMP-9 gelatinolytic activity was evident in rheumatoid arthritis (RA) and GA samples, but no activity was observed in osteoarthritis (OA) samples. Among the 53 SF samples (9 RA, 24 GA, and 20 OA) analyzed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) antigen levels by ELISA, MMP-9 antigen levels were elevated tenfold in GA SF compared with OA SF. In addition, GA synovial tissue extracts revealed elevated levels of MMP-9 expression as compared to OA tissue extracts by Western blot and RT-PCR analysis. Immunohistochemical studies demonstrated that MMP-9 immunoreactivity was more intense in GA than in OA synovial tissues. Furthermore, macrophages activation by gouty crystals in vitro was examined. Crystals stimulated MMP-9 gene expression in macrophage cell line and such stimulation was suppressed by PD98059. These findings suggest that the abnormal production of MMP-9 by macrophages is a reflection of the pathological conditions in joints of patients with GA, and that the activation of MMP-9 in the joint is known to play an important role in joint disease.     

Effect of indomethacin on tumor-infiltrating lymphocytes of a spontaneously developed murine mammary adenocarcinoma

Summary The effect of indomethacin on tumor-infiltrating lymphocytes (TIL) was investigated in a spontaneously developed and weakly immunogenic murine mammary adenocarcinoma (designated JC) in syngeneic immunocompetent BALB/c mice, a tumor model mimicking human disease. Unlike other chemically and virally induced tumors, the expansion of TIL was only possible with an enriched population of lymphocytes, isolated on a discontinuous density gradient then cultured in complete medium containing recombinant human interleukin-2 (rIL-2). The freshly isolated TIL exhibited no cytotoxicity against either the natural-killer-sensitive YAC-1 or the natural-killer-resistant JC cell lines. After culture in rIL-2, the TIL of the JC tumor lysed both YAC-1 and JC. The cytotoxicity of the TIL reached a maximum between the 2nd and 3rd week of culture and decreased thereafter. Antibody-and complement-depletion tests revealed that the cells bearing asialo-GM1 antigen represented the major precursor cells of the cytotoxic TIL, which may explain its nonspecific cytotoxicity. Indomethacin was shown to accelerate the cell proliferation of the rIL-2-activated TIL, but only in the initial 2 weeks of culture and not in later culture. The addition of indomethacin to the rIL-2-containing medium at the beginning of culture resulted in a fast-acting and long-lasting enhancement in cytotoxicity. These results provided a basis for the clinical use of indomethacin, i.e. acceleration in proliferation and augmentation in cytotoxicity. However, the addition of indomethacin at the end of the fourth week after rIL-2 culturing produced neither accelerated proliferation nor augmented cytotoxicity. This study also suggested that a prolonged administration of indomethacin may not be advantageous in clinical trials, since the long-term continuous presence of indomethacin in the culture has resulted in a negative effect on the growth of TIL.     

An automatic segmentation and classification framework for anti-nuclear antibody images

Autoimmune disease is a disorder of immune system due to the over-reaction of lymphocytes against one's own body tissues. Anti-Nuclear Antibody (ANA) is an autoantibody produced by the immune system directed against the self body tissues or cells, which plays an important role in the diagnosis of autoimmune diseases. Indirect ImmunoFluorescence (IIF) method with HEp-2 cells provides the major screening method to detect ANA for the diagnosis of autoimmune diseases. Fluorescence patterns at present are usually examined laboriously by experienced physicians through manually inspecting the slides with the help of a microscope, which usually suffers from inter-observer variability that limits its reproducibility. Previous researches only provided simple segmentation methods and criterions for cell segmentation and recognition, but a fully automatic framework for the segmentation and recognition of HEp-2 cells had never been reported before. This study proposes a method based on the watershed algorithm to automatically detect the HEp-2 cells with different patterns. The experimental results show that the segmentation performance of the proposed method is satisfactory when evaluated with percent volume overlap (PVO: 89%). The classification performance using a SVM classifier designed based on the features calculated from the segmented cells achieves an average accuracy of 96.90%, which outperforms other methods presented in previous studies. The proposed method can be used to develop a computer-aided system to assist the physicians in the diagnosis of auto-immune diseases.     

The Clinical Efficacy and Cardiotoxicity of Fixed-Dose Monthly Trastuzumab in HER2-Positive Breast Cancer: A Single Institutional Analysis

Objective

Trastuzumab-containing treatment regimens have been shown to improve survival outcomes in HER2-positive breast cancer (BC). It is much easier to infuse a fixed one-vial dose to every patient on a regular schedule in the general clinical setting. The aims of this study were evaluating the efficacy of a 440 mg fixed-dose of trastuzumab administered on a monthly infusion schedule, and the risk factors for cardiac events.

Patients and methods

We retrospectively reviewed data from 300 HER2-positive BC patients in our institute: 208 were early-stage BC patients undergoing adjuvant trastuzumab treatment, and 92 were metastatic BC patients treated with trastuzumab infusions until disease progression. There were 181 patients receiving regular trastuzumab infusions every 3 weeks (Q3W; 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks), and the other 119 patients were treated monthly with a fixed 440 mg dose (QM; fixed 440 mg every 4 weeks).

Results

The medians of progression-free survival (PFS) and overall survival (OS) in the adjuvant setting were not reached in both treatment groups. In the metastatic setting, there was no significant difference between groups in PFS or OS. The median time to significant cardiovascular (CV) dysfunction was 4.54 months. The incidence of congestive heart failure requiring medication in our cohort was 3.4%.

Conclusion

In our study, we found that fixed-dose monthly trastuzumab was feasible and effective. In addition, the CV risk was not higher with the fixed-dose protocol. This treatment modality could lower the cost and was easier to implement in clinical practice. Larger prospective randomized studies with longer-term follow up are needed to confirm our results.     

Characterization of a new spontaneously developed murine mammary adenocarcinoma in syngeneic BALB/c hosts

Summary A mouse mammary tumor cell line, desingated JC, has been established from a spontaneously developed primary adenocarcinoma of an aged virgin female BALB/c mouse. Isoenzyme analyses including glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and peptidase proved that this cell line is of murine origin and devoid of contamination from other species. Karyotyping revealed that the number of chromosome ranged from 26 to 100, with a modal number of 40. Electron microscopic examination detected the presence of tonofilament and desmosomes confirming its epithelial nature. In addition, no type B or C virus particle was detected, although intracysternal A particle was observed occasionally. Tumorigenicity in immunocompetent syngeneic hosts was easily established by s.c., i.p., and i.v. injection of viable JC tumor cells. A very weak immunogenicity of the JC tumor was demonstrated through its immunization-challenging on syngeneic immunocompetent hosts. Although no rejection of JC tumor was noted, a significant prolongation for the incubation period before an obvious and palpable tumor growth was detected between the experimental and the control animals. Development of a concomitant immunity was also detected. The JC tumor represents a valuable murine mammary tumor model which is different from other available models because of its unique origin, absence of virus particles, very weak immunogenicity, and high tumorigenicity in syngeneic hosts. The cell line has been maintained for more than 5 yr and has been used for experimental immunotherapy in our laboratory. This work was supported by a research grant IM-416, awarded by the American Cancer Society, Atlanta, Georgia.