Heterogeneity of chemosensitivity of colorectal adenocarcinoma determined by a modified ex vivo ATP-tumor chemosensitivity assay (ATP-TCA)

Advanced colorectal cancer (CRC) has a poor prognosis with a 5-year survival of only 5% despite treatment with chemotherapeutic agents. Response rate and overall survival varies little between the commonly used single agents, although combinations achieve better outcomes. It is well established that considerable heterogeneity exists between cancers of the same tissue type, but it has been difficult to establish this for CRC. We therefore investigated the heterogeneity of chemosensitivity in CRC using a modified version of the ex vivo ATP-tumor chemosensitivity assay (ATP-TCA) capable of handling infected tumor tissue. Fifty-three specimens of primary solid or malignant effusions of CRC were tested, of which 46 (87%) were evaluable. There were considerable differences in sensitivities between individuals. The most active single cytotoxic agents in the assay were identified as 5-fluorouracil, irinotecan and mitomycin C (MMC). Cells were exposed to combinations of drugs added simultaneously at the same concentrations tested as single agents. All drug combinations achieved greater growth inhibition than drugs used alone. MMC+gemcitabine was found to be the most effective combination in 83% of specimens. The ATP-TCA has previously been shown to be a good predictor of response to chemotherapy in other tissue types. The degree of heterogeneity demonstrated from these results suggests that the ATP-TCA could be used to identify patients who might benefit from specific chemotherapeutic agents alone or in combination.     

Automated measurement of microaneurysm turnover

PURPOSE: An automated system for the measurement of microaneurysm (MA) turnover was developed and compared with manual measurement. The system analyses serial fluorescein angiogram (FA) or red-free (RF) fundus images; fluorescein angiography was used in this study because it is the more sensitive test for MAs. Previous studies have shown that the absolute number of MAs observed does not reflect the dynamic temporal nature of the MA population. In this study, almost half of the MAs present at baseline had regressed after a year and been replaced by new lesions elsewhere. METHODS: Two clinical datasets were used to evaluate the performance of the automated turnover measurement system. The first consisted of 10 patients who had two fluorescein angiograms acquired a year apart. These data were analyzed, both manually and using the automated system, to investigate the inter- and intraobserver variations associated with manual measurement and to assess the performance of the automated system. The second dataset contained FAs from a further 25 patients. This dataset was analyzed only with the automated system to investigate some properties of microaneurysm turnover, in particular the differing detection sensitivities of new, static and regressed microaneurysms. RESULTS: Manual measurements exhibited large inter- and intraobserver variation. The sensitivity and specificity of the automated system were similar to those of the human observers. However, the automated measurements were more consistent-an important condition for accurate turnover quantification. Regressed MAs were more difficult to detect reliably than new MAs, which were themselves more difficult to detect reliably than static MAs. CONCLUSIONS: The automated system was shown to be fast, reliable, and repeatable, making it suitable for processing large numbers of images. Performance was similar to that of trained manual observers.     

双链断裂修复蛋白hKu70缺陷细胞株的建立及其生物学特性

目的 建立并鉴定DNA双链断裂(DSB)修复蛋白hKu70缺陷细胞株,并观察该缺陷细胞的某些生物学效应,用于AKu70基因功能及职业有害因素对DNA双链断裂修复影响的研究。方法 用构建的AKu70基因反义RNA绿色荧光蛋白真核表达载体(pEGFP—CI—K)转染人胚肺成纤维细胞(HLF),用蛋白兔疫印迹法鉴定转染细胞中AKu70基因的表达水平。同时观察转染细胞生长形态,绘制生长曲线,软琼脂培养法鉴定恶性程度。结果 pEGFP—CI—K载体在转染细胞内可较稳定表达,hKu70蛋白缺陷细胞株AKu70基因的蛋白表达水平下降了42％,转染后hKu70蛋白缺陷细胞生长形态、生长速度无明显变化,软琼脂培养未见细胞集落。结论 成功建立和鉴定了hKu70蛋白缺陷细胞株,该缺陷不足以单独引起可观察的某些生物学效应。     

Outcome of ATP-based tumor chemosensitivity assay directed chemotherapy in heavily pre-treated recurrent ovarian carcinoma

Background

We wished to evaluate the clinical response following ATP-Tumor Chemosensitivity Assay (ATP-TCA) directed salvage chemotherapy in a series of UK patients with advanced ovarian cancer. The results are compared with that of a similar assay used in a different country in terms of evaluability and clinical endpoints.

Methods

From November 1998 to November 2001, 46 patients with pre-treated, advanced ovarian cancer were given a total of 56 courses of chemotherapy based on in-vitro ATP-TCA responses obtained from fresh tumor samples or ascites. Forty-four patients were evaluable for results. Of these, 18 patients had clinically platinum resistant disease (relapse < 6 months after first course of chemotherapy). There was evidence of cisplatin resistance in 31 patients from their first ATP-TCA. Response to treatment was assessed by radiology, clinical assessment and tumor marker level (CA 125).

Results

The overall response rate was 59% (33/56) per course of chemotherapy, including 12 complete responses, 21 partial responses, 6 with stable disease, and 15 with progressive disease. Two patients were not evaluable for response having received just one cycle of chemotherapy: if these were excluded the response rate is 61%. Fifteen patients are still alive. Median progression free survival (PFS) was 6.6 months per course of chemotherapy; median overall survival (OAS) for each patient following the start of TCA-directed therapy was 10.4 months (95% confidence interval 7.9–12.8 months).

Conclusion

The results show similar response rates to previous studies using ATP-TCA directed therapy in recurrent ovarian cancer. The assay shows high evaluability and this study adds weight to the reproducibility of results from different centres.     

Persistence of acanthamoeba antigen following acanthamoeba keratitis

AIM: To investigate the hypothesis that persistent corneal and scleral inflammation following acanthamoeba keratitis is not always caused by active amoebic infection but can be due to persisting acanthamoebic antigens METHODS: 24 lamellar corneal biopsy and penetrating keratoplasty specimens were obtained from 14 consecutive patients at various stages of their disease and divided for microscopy and culture. Histological sections were immunostained and screened for the presence of Acanthamoeba cysts by light microscopy. Cultures were carried out using partly homogenised tissues on non-nutrient agar seeded with E coli. Clinical data were obtained retrospectively from the case notes of these patients. RESULTS: Of the 24 specimens, 20 were obtained from eyes that were clinically inflamed at the time of surgery. Acanthamoeba cysts were present in 16 (80%) of these 20 specimens, while only five (25%) were culture positive. Acanthamoeba cysts were found to persist for up to 31 months after antiamoebic treatment. CONCLUSION: These findings support the hypothesis that Acanthamoeba cysts can remain in corneal tissue for an extended period of time following acanthamoeba keratitis and may cause persistent corneal and scleral inflammation in the absence of active amoebic infection. In view of these findings, prolonged intensive antiamoebic therapy may be inappropriate when the inflammation is due to retained antigen rather than to viable organisms     

Demonstration of biofilm in infectious crystalline keratopathy using ruthenium red and electron microscopy

OBJECTIVE: Bacterial biofilm formation has been implicated in the pathogenesis of infectious crystalline keratopathy. Biofilm cannot be visualized by electron microscopy without the addition of a fixative that stabilizes the polysaccharide-rich bacterial extracellular matrix that surrounds the bacterial colonies in a biofilm. We used ruthenium red as a fixative to evaluate corneal biopsy specimens for the presence of bacterial biofilm in three cases of infectious crystalline keratopathy (ICK) and five cases of chronic microbial keratitis without crystalline changes. DESIGN: Case series with clinicopathologic correlation. PARTICIPANTS: Eight patients underwent corneal biopsy or therapeutic keratoplasty as part of their management for chronic unresponsive microbial keratitis. METHODS: The corneal specimens removed were trisected for microbiology, pathology, and transmission electron microscopy (TEM). The TEM specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% ruthenium red. MAIN OUTCOME MEASURES: Demonstration of bacterial biofilm with TEM. RESULTS: TEM demonstrated organisms with a surrounding extracellular matrix consistent with a bacterial biofilm in the three cases of ICK but not in the five other cases of chronic microbial keratitis. CONCLUSIONS: The presence of biofilm in ICK can be demonstrated with TEM with appropriate fixation techniques that stabilize the bacterial extracellular matrix. Biofilm stains intensely with periodic acid-Schiff because of the polysaccharide-rich extracellular matrix and weakly with Gram stain because of the high proportion of nonviable organisms. Biofilm formation occurs in ICK but probably not in chronic bacterial keratitis without crystalline changes. Secretion of an extracellular matrix by bacteria to form a biofilm is a response to a nutrient-deprived environment in which growth and replication is depressed. The extracellular matrix of the biofilm may mask bacterial antigens, explaining the relative lack of inflammatory response in these infections. It may also be one of the mechanisms explaining the resistance to in vivo antimicrobial therapy when in vitro sensitivities have been proven.     

Studies of cell death (apoptosis) and cell division in leprosy granulomas

We have studied the histological changes across leprosy lesions by taking biopsies from the center and edge of the lesions and from the clinically uninvolved skin outside the lesions. A comparison of the granuloma fraction (GF) between biopsies from the center and edge of lesions and the adjacent unremarkable skin shows that the greatest GF is found at the edge of lesions, except in early tuberculoid (BT) cases when biopsies from the center have the greatest GF. Central healing of leprosy lesions occurs without tissue necrosis or appreciable fibrosis. Apoptosis, a form of individual cell death in living tissues, is known to be the mechanism of cell loss in a variety of situations, and we have found it to occur in leprosy lesions. Apoptotic activity is greatest at the edge of established tuberculoid lesions, but can be found in the center of the lesion in early cases. We, therefore, suggest that apoptosis is the mechanism by which epithelioid cells are lost during central healing in tuberculoid leprosy lesions. In the small number of multibacillary cases studied, apoptosis were found in biopsies from both the center and edge of the lesions. Mitoses can be found in biopsies from both lepromatous and tuberculoid lesions. However, the degree of mitotic activity does not appear to be related to the position of the biopsy within the lesion, and immigration of monocytes into the granulomas may be of greater importance than cell division in maintaining the numbers of epithelioid cells or macrophages present.     

Variations in treatment of femoral neck fractures in Alberta.

OBJECTIVES: To examine, in the province of Alberta, temporal trends, regional variations in treatment options and in-hospital death rates after a femoral neck fracture. DESIGN: A retrospective cohort study. PATIENTS: Six years' data were abstracted from the Alberta Morbidity File, the Alberta Health Stakeholder File and the Alberta Health Care Claims File. Patients were included if they were Alberta residents, aged 65 years or older, had sustained a femoral neck fracture and had undergone internal fixation, hemiarthroplasty or total hip arthroplasty. MAIN OUTCOME MEASURES: Death rates, arthroplasty rates and hospital stay. RESULTS: In-hospital death rates were similar across hospitals, with risks being higher for men, patients aged 80 years or older and those with more comorbid conditions. Arthroplasty rates varied from 58% to 77% among hospitals, and hospital stays associated with arthroplasty were significantly longer than those associated with internal fixation. The chance of undergoing arthroplasty varied from hospital to hospital by gender and by the number of comorbid conditions. CONCLUSION: Regional variations suggest lack of agreement among Alberta's surgeons as to how best to treat femoral neck fractures.