DNA in situ sensitivity to denaturation: a new parameter for flow cytometry of normal human colonic epithelium and colon carcinoma

Modal DNA (ploidy) and sensitivity of DNA in situ to denaturation by acid have been analyzed by flow cytometry of 10 colorectal adenomas and 35 adenocarcinomas; 39 normal mucosa samples served as controls. A new method was developed to denature DNA in chromatin of the freshly isolated, intact, and unfixed individual cell nuclei from surgically resected material. The sensitivity of DNA denaturation (T alpha) was assayed by metachromatic staining with acridine orange and calculated as a ratio of the alpha t index of the tumor sample to the alpha t index of normal mucosa; the alpha t index is that fraction of DNA, following treatment at pH 1.4, that stains metachromatically with acridine orange at pH 2.6. All adenomas were diploid and in nine of 10 the T alpha value was close to 1.00, indicating no difference from control specimens in DNA sensitivity to denaturation. Forty-nine% of adenocarcinomas were aneuploid. Forty-six% of adenocarcinomas differed from normal in sensitivity of DNA to denaturation; the T alpha value was lower than 0.90 indicating that chromatin of the tumor cells was more resistant to denaturation than control cells. There was no correlation between sensitivity to denaturation of DNA and incidence of aneuploidy. However, there was a correlation between T alpha and the pathologically determined stage of disease. There was increased resistance to denaturation in 58% of tumors classified as Dukes' C/D stage, in 36% of tumors classified as Dukes' B, and in 20% classified as Dukes' A stage of the disease. Statistical analysis of these results revealed significant differences between distributions of T alpha in noninvasive (Adenomas and Dukes' A) versus invasive (Dukes' B and C/D) tumors with level of significance at P = 0.02. The data suggest that acid denaturation of DNA in situ may be a valuable adjunct in assessing the biology of colon cancer. The molecular basis for this phenomenon is discussed.     

DNA in situ sensitivity to denaturation in bladder cancer and its correlation with tumor stage

DNA content and sensitivity of DNA in situ to denaturation by acid were analyzed by flow cytometry of cell nuclei freshly isolated from the bladder tumors of 32 patients and were compared with normal urothelium of 8 subjects. DNA sensitivity to denaturation was assessed in RNase treated cells by acridine orange metachromasia following partial denaturation with hydrochloric acid; the extent of denatured DNA is given as an index (alpha t), representing the ratio of single stranded to total DNA per nucleus. Of the low stage tumors (papillomas, Ta, Tis, T1) 11 of 18 (61%) were aneuploid. Of the high stage tumors (T2 and T3a) 11 of 14 (79%) were aneuploid. DNA in nuclei of normal transitional epithelium was very sensitive to denaturation, as was papilloma, characterized by nuclear alpha t indices of 0.73 +/- 0.01 (SD) and 0.73 +/- 0.04, respectively. Nuclear DNA of noninvasive carcinomas (Ta, Tis) was significantly more resistant to denaturation (alpha t = 0.69), and DNA of invasive carcinomas was most resistant, ranging from alpha t = 0.61 (T1 tumors) to alpha t = 0.59 (T2 tumors) to alpha t = 0.57 (T3 tumors). High stage tumors as a group (T2, T3) had significantly different (lower) alpha t values than low stage tumors (Ta, Tis, T1). In model cell culture systems it is known that a decrease in alpha t index, i.e., greater resistance to denaturability, occurs as cells transit from resting phase into the cell cycle. Whether the alpha t index can be used to estimate resting vesus cycling cells of human tumors is still speculative; changes in DNA denaturability also are known to occur with changes in chromatin structure during cell differentiation and in transformation. However, the empirical relationship between alpha t index and tumor stage, of itself, may prove clinically useful in identifying more advanced and perhaps more aggressive tumors.     

运用原位DNA末端标记法研究细胞凋亡对乳腺癌预后的影响

研究临床乳腺癌标本中细胞凋亡的发生情况,评价其在预测后在的意义。方法搜集９１例浸润性乳腺癌的石蜡组织切片,运用原位ＤＮＡ断裂位点的末端标记法,检测细胞凋亡,计算凋亡细胞占肿瘤细胞的百分比,求得凋亡指数。结果本组病例细胞凋亡发生率为９１．２％,凋亡指数分为两组：０～０．２１和０．２８～０．６２,凋亡指数高低与腋淋巴结转称相关（Ｐ＜０．０１）。生存率单因素分析中,凋亡指数高的病例,无病生存率（Ｐ＝０．００９５）及总生存率（Ｐ＝０．０３４８）均优于凋亡指数低的病例。但Ｃｏｘ模型多因素分析末能指示凋亡指数是一个独立的预后指标。结论在乳腺癌组织中,细胞凋亡是一种自发现象,其发生情况各有不同,研究初步提示了细胞凋亡在乳腺癌预后中的作用,了解了凋亡和腋淋巴结转移的相关性。     

Mitotic rate,DNA distribution,and chromatinin situ sensitivity to heparin in breast cancer

Summary The purpose of this study was to characterize breast carcinomas by cell kinetic parameters. Mitotic rate (MR) and flow cytometrically (FCM) measured cell cycle distribution as well as chromatin testing in situ employing heparin for determination of activated chromatin, provided the following results:MR counted in 73 unselected carcinomas showed an increase up to a tumor size of 4.2cm (p < 0.05); beyond this diameter, the MR was found to decrease.In T1-T2 carcinomas, cell cycle stage analysis yielded higher percentages of cells in S and G₂M phase for ductal (13% and 12%, N = 22) than for lobular (8% and 7%, N = 8) node-negative carcinomas (p < 0.002). In ductal carcinomas, lymph node involvement was reflected by higher % G₂M values (15%, N = 26) compared with negative cases (12%, N = 22) (p < 0.05).Ductal node-positive T3-T4 carcinomas (N = 10) revealed a higher % S value (16%) than their T1-T2 counterparts. A correlation between MR and % G₂M was established only up to a tumor size of 4.2 cm (r = 0.39, p < 0.05).A highly sensitive (H) and a poorly sensitive (P) subgroup of carcinomas with respect to heparininduced changes in fluorescence intensity of the G_1/0 peak of the DNA aneuploid cell line were identified, as previously shown [1]. These subgroups were here updated with a larger number of carcinomas and were limited to T1-T2 cancers (N = 57). Group H included more younger patients (p < 0.005), less cases with nodal involvement in ductal carcinomas (p < 0.05), and lower % G₂M values in lobular node-negative cases (p < 0.05), than group P. DNA diploid cells always existing in DNA aneuploid carcinomas are more sensitive than their aneuploid counterparts (p < 0.01); however, they strengthen the stratification to H and P. We suggest H carcinomas to be less aggressive than P carcinomas. Small breast carcinomas are recommended to cell kinetic investigations for individualizing adjuvant therapy.     

Xenografts in pharmacologically immunosuppressed mice as a model to test the chemotherapeutic sensitivity of human tumors

A human tumor xenograft model using pharmacologically immunosuppressed mice was assessed for its suitability to test preclinically the sensitivity of colorectal carcinomas, bone sarcomas and melanomas against anticancer agents. Besides ionizing radiation, 14 cytotoxic drugs including 5-fluorouracil (5-FU), dimethylmyleran (DMM), cytosine arabinoside, cyclophosphamide, melphalan, BCNU, mitomycin C, adriamycin, bleomycin, etoposide, vinblastine, cisplatin, procarbazine and DTIC were assayed. Ionizing radiation, 5-FU and DMM were also applied at lethal doses followed by bone-marrow rescue heavy therapy. Four colon carcinomas responded poorly to most of the agents but one tumor displayed marked sensitivity to BCNU. Lethal doses of radiation, 5-FU and DMM could also show considerable activity. High sensitivity was shown by a Ewing sarcoma to DMM and cyclophosphamide and by an osteosarcoma to the latter drug. No strong effects were seen against melanomas. Lethal doses of DMM induced the best regression of one colon carcinoma. In general, the superiority of heavy therapy for solid human tumors compared to maximally tolerated doses was demonstrated. Individual carcinomas of the same type displayed different drug sensitivity.     

Zinc-alpha2-glycoprotein expression as a marker of differentiation in human oral tumors.

Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.     

Intratumoral oestrone sulphatase activity as a prognostic marker in human breast carcinoma.

Oestrone sulphatase is an important source of local synthesis of biologically active oestrogens in human breast cancer. The oestrone sulphatase enzyme in the particulate fraction of human breast carcinoma was characterised. The Km was 8.91 microM, and the Vmax was 0.022 nmol min-1 mg-1. Oestrone sulphatase activity was detected in 93 of 104 human breast carcinoma samples (89%), and mean activity was 0.041 nmol min-1 mg-1 (range 0-0.399 nmol min-1 mg-1). There was no significant correlation between intratumoral oestrone sulphatase activity and oestrogen receptor status, or with any other prognostic factors. Intratumoral enzyme levels were not associated with time to recurrence or with overall survival time. It thus appears that, although a useful source of intratumoral oestrogens, oestrone sulphatase activity is not of prognostic significance in breast carcinoma.     

Thyroxine-binding globulin as a marker of liver tumors

This investigation was undertaken to evaluate the level of thyroxine-binding globulin (TBG) as a marker of liver tumors. TBG examination was performed on 42 patients with primary hepatocellular carcinoma (PHC) and 50 postoperative patients with metastases to the liver. alpha-Fetoprotein (AFP) and carcinoembryonic antigen (CEA) concentrations were determined at the same time. AFP was positive in 90.5% of the patients with PHC, and TBG was higher than normal in 69% of patients with PHC. In the 50 patients with liver metastasis, the TBG was higher than normal in 72%, and CEA was also increased in 64%. CEA was negative in 18 of 50 patients with liver metastasis. On the other hand, of the 18 CEA-negative patients, 14 (77.8%) had a higher than normal TBG concentration. This was a significant, but not specific, increase. Therefore, elevated TBG levels are a sensitive, although nonspecific, tumor marker to determine the presence of liver tumors, especially in cases of metastasis to the liver.     

Serum interferon as a biological marker in malignant tumors

A total of 745 serum interferon (IFN) determinations were carried out on the serum of 332 cancer patients with a variety of tumors. Disease activity was defined as either 'active' or 'nonactive' as based on clinical examination, the results of laboratory tests and radiological criteria. IFN levels were estimated for both disease activity and mode of treatment. A statistically significant higher level of IFN in the serum of active, as compared to that of nonactive disease, was established. Furthermore, a slight increase in the IFN level was observed in patients before initiating chemotherapy as compared to untreated patients, followed by a decrease in those patients undergoing chemotherapy. These differences between groups were not found to be statistically significant. However, in the cases of rectum, prostate and female genital cancers, a significant elevated level of serum IFN was observed at a late stage of the disease, followed by a decrease after chemotherapy treatment. In addition, a significant lower level of serum IFN was detected in breast cancer patients shortly after radiotherapy as compared to those before radiotherapy. We conclude that IFN, known to play a role in the control mechanisms of developing malignant processes, can be determined in the serum and used to monitor disease activity in individual patients. The inhibition of IFN release depends in part on therapy and can be used for monitoring treatment schedules along the course of disease.     

Collagenase-3 expression in breast myofibroblasts as a molecular marker of transition of ductal carcinoma in situ lesions to invasive ductal carcinomas

Collagenase-3 (matrix metalloproteinase 13; MMP-13), a protease originally identified in breast carcinoma, is characterized by a potent degrading activity against a wide spectrum of extracellular matrix proteins. The aims of this study were to localize and identify the MMP-13-expressing cells in invasive human breast carcinoma and to evaluate the role of MMP-13 in transition to invasive lesions by studying ductal carcinoma in situ (DCIS). We found expression of MMP-13 in stromal fibroblast-like cells in all 21 invasive ductal carcinomas studied and in 4 of 9 invasive lobular carcinomas. In most carcinomas, expression of MMP-13 was limited to small stromal foci in the tumor area. Combined in situ hybridization and immunohistochemistry showed coexpression of alpha-smooth muscle actin immunoreactivity and MMP-13 mRNA in myofibroblasts. In contrast, cytokeratin-positive cancer cells, alpha-smooth muscle actin-positive vascular smooth muscle cells, CD68-positive macrophages, and CD31-positive endothelial cells were all MMP-13 mRNA negative. In situ hybridization for MMP-13 in 17 DCIS lesions revealed expression in 10 cases. Immunohistochemical analysis of all DCIS cases identified microinvasion in 8 of the 17 lesions. Seven of the eight lesions with microinvasion were MMP-13 positive. Further analysis showed that MMP-13 expression was often associated with the microinvasive events. This particular expression pattern was unique for MMP-13 among other MMPs analyzed, including MMP-2, -11, and -14. We conclude that MMP-13 is primarily expressed by myofibroblasts in human breast carcinoma and that expression in DCIS lesions often is associated with microinvasive events. On the basis of these data, we propose that MMP-13 may play an essential role during transition of DCIS lesions to invasive ductal carcinomas.     

A viral antigen as a marker for the prognosis of human breast cancer

An antigen present in human breast tumor cells, and which is immunologically related to the envelope protein (gp52) of murine mammary tumor virus, was used as a marker for the detection of breast cancer in an Israeli population. The results show that the antigen was detectable in 128 of 204 breast carcinomas tested (62.7%). The immunological reaction was not detected in normal breast tissue, benign breast tumors, ductal hyperplasia or in primary malignancies in other organs. A significantly higher percentage of cases with demonstrable antigen was found in Israeli women born in North Africa (78%) as compared to women of European origin (60.6%). The frequency of detection of the antigen was higher in stage IV (80%) as compared to stage I (15%), suggesting that the gp52 cross-reacting antigen is a marker for the severity of the disease. Moreover, a retrospective study of 97 cases of stage II breast cancer shows that if the antigen is detected at the time of mastectomy, one can usually predict an unfavorable prognosis. Survival data analysis indicates that patients without detectable antigen survived significantly longer than those with a detectable antigen.     

The feasibility of circulating tumor DNA analysis as a marker of recurrence in triple-negative breast cancer

Triple-negative breast cancer (TNBC) has a poorer prognosis than other breast cancer subtypes; therefore, identifying markers of early recurrence is important. The present study aimed to establish a liquid biopsy protocol for droplet digital PCR-based detection of frequently mutated genes in patients with TNBC. Tumor DNA from 36 patients with TNBC who relapsed within 2 years after surgical resection was retrospectively analyzed. Somatic mutational profiles were evaluated using targeted sequencing to identify frequently mutated genes and genes associated with molecularly targeted therapies. The association between genetic alterations and associated protein phosphorylation was investigated using immunohistochemical analysis. Recurrent hot spot mutations in the plasma were monitored over time. Mutation-specific probes were used to successfully detect mutations in the blood samples of patients who were positive for PIK3CA H1047R and AKT1 E17K mutations. Somatic mutations in AKT1 (14.9%) and PIK3CA (25.5%) were frequently identified in the data. Robust phosphorylation of AKT and S6RP was more common in tumors with PIK3CA H1047R and AKT1 E17K mutational background than in tumors with wild-type PIK3CA and AKT1. In conclusion, the present study evaluated a high-sensitivity detection system for frequently mutated genes that was also applicable for cell-free DNA. The PI3K/AKT pathway was revealed to be activated in patients harboring PIK3CA H1047R and AKT1 E17K mutations; therefore, the PI3K/AKT pathway may be a promising candidate for targeted therapy in these patients.     

Plasminogen activator as a functional marker for estrogen dependence in human breast cancer cells

To examine whether plasminogen activator reflects the functional state of estrogen receptors in human breast cancer, the enzyme activities were determined in extracts prepared from 160 breast cancer specimens and compared on qualitative and quantitative bases with the levels of steroid receptors, such as cytoplasmic estrogen receptor (ERC), progesterone receptor (PgR) and nuclear estrogen receptor (ERN). With any receptor, plasminogen activator activity was significantly higher in receptor-positive tumors than in receptor-negative tumors. When these breast tumors were categorized into 8 groups in terms of combinations of receptor status, breast cancers which were positive for all these receptors were found to contain the highest plasminogen activator activity. Furthermore, quantitative analyses demonstrated positive correlations of the enzyme activity with either ERC content (correlation coefficient +0.37, P less than 0.001) or PgR content (correlation coefficient +0.45, P less than 0.001). These results strongly suggest that plasminogen activator can be used as an effective functional marker for hormone dependence in human breast cancer.     

DNA methylation marker to estimate the breast cancer cell fraction in DNA samples

Estimation of the cancer cell fraction in breast cancer tissue is important for exclusion of samples unsuitable for multigene prognostic assays and a variety of molecular analyses for research. Here, we aimed to establish a breast cancer cell fraction marker based on DNA methylation. First, we screened genes unmethylated in non-cancerous mammary tissues and methylated in breast cancer tissues using microarray data from the TCGA database, and isolated 12 genes. Among them, four genes were selected as candidate marker genes without a high incidence of copy number alterations and with broad coverage across patients. Bisulfite pyrosequencing analysis of additional breast cancer biopsy specimens purified by laser capture microdissection (LCM) excluded two genes, and a combination of SIM1 and CCDC181 was finally selected as a fraction marker. In further additional specimens without LCM purification, the fraction marker was substantially methylated (≥?20%) with high incidence (50/51). The cancer cell fraction estimated by the fraction marker was significantly correlated with that estimated by microscopic examination (p?<?0.0001). Performance of a previously established marker, HSD17B4 methylation, which predicts therapeutic response of HER2-positive breast cancer to trastuzumab, was improved after the correction of cancer cell fraction by the fraction marker. In conclusion, we successfully established a breast cancer cell fraction marker based on DNA methylation.