Assessing acute myeloid leukemia susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CCCTC-binding factor/cohesin binding sites.

An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of nonfusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to nonfusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin-binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in nonfusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons.

Circulating Bone-related Markers and YKL-40 Versus HER2 and TOPO2a in Bone Metastatic and Nonmetastatic Breast Cancer: Diagnostic Implications.

BACKGROUND: The bone represents one of the most common sites of metastases in breast cancer. The aim of the current study was to evaluate the diagnostic potential of several circulating markers to detect metastasis to bones in patients with breast cancer. PATIENTS AND METHODS: Receptor activator of Nuclear Factor-kappa ß (NF-Kß) ligand (RANKL), osteoprotegrin (OPG), vitamin D (VIT D), Chitinase-3-like protein 1; also known as YKL-40, topoisomerase IIα (TOPO2a), and human epidermal growth factor receptor 2 (HER2) were measured in blood samples obtained from 122 patients with breast cancer and 25 healthy controls. RESULTS: All biomarkers were significantly elevated in patients with breast cancer with bone metastasis compared with nonmetastatic patients except YKL-40. RANKL had the highest diagnostic performance for bone metastasis detection with an area under the curve of 97.3, a sensitivity of 85%, and a specificity of 98.6%. Furthermore, logistic regression analysis resulted in a model of RANKL combined with HER2 that had even higher discriminatory power of metastasis to bones than that of RANKL alone. Overall correct classification of the model was 98.9%. CONCLUSION: We recommend that measuring RANKL together with HER2 can be routinely applied to allow early detection of bone metastases in patients with breast cancer.

Topoisomerase IIα as a prognostic factor in pituitary tumors.

INTRODUCTION: There is an ongoing search for markers of pituitary tumor proliferation and progression that could facilitate further treatment and patient monitoring. OBJECTIVES: We studied topoisomerase IIα (topo IIα) expression in different types of pituitary adenomas to evaluate its prognostic value. PATIENTS AND METHODS: In a retrospective study of 60 patients (mean age, 46.7 ±17.6 y) who underwent pituitary tumor surgery, expression of topo IIα was assessed by immunohistochemistry and compared with histopathological tumor features, clinical symptoms, magnetic resonance imaging, and postoperative tumor recurrence or progression. RESULTS: Expression of topo IIα was observed in 44 of 60 pituitary adenomas (73%). The highest topo IIα index was observed in adrenocorticotropic hormone (ACTH)-secreting tumors (median, 1.13% [0.37-1.21]), followed by silent-ACTH tumors (0.94% [0.89-1.0]), and hormone immunonegative adenomas (0.8% [0.65-1.55]). There were no differences in topo IIα expression with respect to age or sex. Significant correlations were observed between the topo IIα index and tumor size, its invasiveness, abnormal ocular test results, and postoperative tumor recurrence. In patients with a topo IIα index exceeding 1%, we observed a 3.5-fold higher relative risk of tumor recurrence as compared with patients with a topo IIα index lower than 1% (95% confidence interval: 1.8-6.9; P <0.001). Patients with acromegaly who received somatostatin analogues before the surgery had a lower median topo IIα index compared with untreated patients (0%[0-0.22] vs. 0.71% [0.17-1.0]; P <0.05). CONCLUSIONS: In our study group, a topo IIα index exceeding 1% was a prognostic factor for tumor recurrence or progression, especially in patients with hormonally inactive adenomas, which facilitates patient selection for intensive postoperative treatment. Use of somatostatin analogues in acromegaly inhibits topo IIα expression, providing molecular evidence for the effectiveness of these analogues.

Phase I dose escalation study with irinotecan, capecitabine, epirubicin, and granulocyte colony-stimulating factor support for patients with solid malignancies.

OBJECTIVES: Preclinical studies using sequences of topoisomerase I and II inhibitors suggested synergism; preliminary clinical studies, resulting in enhanced antitumor responses, confirm this in selected malignancies. This study determined the maximum-tolerated dose (MTD), toxicity, and pharmacokinetics of irinotecan (CPT-11), capecitabine, and epirubicin in patients with metastatic adenocarcinoma of lung, breast, or gastrointestinal tract. Correlation of topoisomerase IIbeta was also done. METHODS: Eligibility criteria included the following: documented adenocarcinoma of the lung, breast, or gastrointestinal tract, <3 prior chemotherapy regimens, Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2, age > or =18 years, adequate organ function, and signed informed consent. Irinotecan was administered at 250 mg/m2 intravenously day 1 every 21-day cycle, but was reduced to 180 mg/m2 in cohort 2 due to toxicity. Capecitabine was administered at 750 mg/m2 twice daily days 2 to 14 in cohort 1 but only on days 2 to 7 from cohort 2 due to early neutropenia and to allow for prophylactic granulocyte colony-stimulating factor (GCSF) support. Epirubicin was administered at 40 mg/m2 in cohort 1, but reduced to 30 mg/m2 in cohort 2, then reescalated until the MTD was reached. RESULTS: Toxicity was assessed in 21 patients; response was assessed in 17 patients. The most common grade 3 to 4 toxicities included neutropenia (57.1%) and anemia (28.6%). The MTD of epirubicin was 50 mg/m2. In evaluable patients, there were 2 partial responses (11.8%) and 13 stable disease (76.5%); these correlated well with topoisomerase IIbeta. CONCLUSIONS: The recommended doses for phase II studies: irinotecan 180 mg/m2 day 1, epirubicin 50 mg/m2 day 2, and capecitabine 750 mg/m2 twice daily days 2 to 7 of each 21-day cycle. This combination is reasonably active and warrants evaluation in the phase II setting.

Topoisomerase II beta expression level correlates with doxorubicin-induced apoptosis in peripheral blood cells.

Anthracyclines are widely used in oncology. Both the response and side-effects of anthracyclines are individually variable, but determinants or predictive markers of this variability are not available. We investigated the variability in the expression of the anthracycline targets topoisomerases II (topo II) alpha and beta and its significance for the apoptotic response following exposure to the anthracycline doxorubicin. Only topo II beta protein expression was detected in peripheral blood cells. Usually considered a constitutively expressed protein, topo II beta varied 3-, 18-, and 16-fold on the mRNA, protein and activity levels, respectively, among the volunteers tested. In addition, the expression of topo II beta was modified by several mitogens, suggesting a role in the regulation of cell cycle. Strikingly, topo II beta activity correlated statistically significantly with the apoptotic response in peripheral blood leukocytes exposed to 1 microM doxorubicin. A longitudinal study in a subset of study subjects demonstrated that 30% of the topo II expression variability may be inherited. However, resequencing of the TOP2B gene in 48 unrelated individuals revealed only 8 gene variants, none of them with obvious effects on the expression or protein sequence of topo II beta. Taken together, the apoptotic response to doxorubicin in peripheral blood cells may be mediated by topo II beta. The expression level of topo II beta is intra- and inter-individually variable, and may in part determine the apoptotic response to doxorubicin and other anthracyclines.

Sequential oral 9-nitrocamptothecin and etoposide: a pharmacodynamic- and pharmacokinetic-based phase I trial.

PURPOSE: Resistance to topoisomerase (topo) I inhibitors has been related to down-regulation of nuclear target enzyme, whereas sensitization to topo II inhibitors may result from induction of topo II by topo I inhibitors. Here, we evaluated a sequence-specific administration of a topo I inhibitor followed by a topo II inhibitor. EXPERIMENTAL DESIGN: Twenty-five patients with advanced or metastatic malignancies were treated with increasing doses (0.75, 1.0, 1.25, 1.5, 1.75, or 2.0 mg/m(2)) of 9-nitrocamptothecin (9-NC) on days 1 to 3, followed by etoposide (100 or 150 mg/d) on days 4 and 5. At the maximally tolerated dose, 20 additional patients were enrolled. The median age was 60 years (range, 40-84 years). Endpoints included pharmacokinetic analyses of 9-NC and etoposide, and treatment-induced modulations of topo I and II expression in peripheral blood mononuclear cells. RESULTS: Neutropenia, thrombocytopenia, nausea, vomiting, diarrhea, and fatigue were dose-limiting toxicities and occurred in six patients. Despite a median number of four prior regimens (range 1-12), 2 (4%) patients had an objective response and 13 (29%) patients had stable disease. In contrast to the expected modulation in topo I and IIalpha levels, we observed a decrease in topo IIalpha levels, whereas topo I levels were not significantly altered by 9-NC treatment. CONCLUSIONS: Sequence-specific administration of 9-NC and etoposide is tolerable and active. However, peripheral blood mononuclear cells may not be a predictive biological surrogate for drug-induced modulation of topo levels in tumor tissues and should be further explored in larger studies.

Pharmacokinetically guided phase I trial of topotecan and etoposide phosphate in recurrent ovarian cancer.

A pharmacokinetically guided phase I study of topotecan and etoposide phosphate was conducted in recurrent ovarian cancer. The scheduling of the topoisomerase I and II inhibitors was determined using in vitro activity data. All patients had recurrent disease following prior platinum-containing chemotherapy. Patients had a World Health Organisation performance status of 0-2 and adequate bone marrow, renal and hepatic function. Treatment was with topotecan intravenously for 5 days followed immediately by a 5-day intravenous infusion of etoposide phosphate (EP), with pharmacokinetically guided dose adjustment. Plasma etoposide levels were measured on days 2 and 4 of the infusion. A total of 21 patients entered the study. In all, 48% were platinum resistant and 71% had received prior paclitaxel. The main toxicities were haematological, short lived and reversible. A total of 29% of patients experienced grade 4 thrombocytopenia and 66% grade 4 neutropenia after the first cycle. Neutropenia and thrombocytopenia was dose limiting. The maximum-tolerated dose was topotecan 0.85 mg m(-2) day(-1) days 1-5 followed immediately by a 5-day infusion of EP at a plasma concentration of 1 mug ml(-1). The response rate (RR) was 28% in 18 evaluable patients. There was marked interpatient variability in topoisomerase IIalpha levels measured from peripheral lymphocytes, with no observed increase following topotecan. This regimen of topotecan followed by EP demonstrated good activity in recurrent ovarian cancer and was noncrossresistant with paclitaxel. Both the toxicity and RR was higher than would be expected from the single agent data, in keeping with synergy of action.

Topoisomerase IIalpha and APE/ref-1 are associated with pathologic response to primary anthracycline-based chemotherapy for breast cancer.

The aim of this study was to evaluate the role of several biological and histological markers (topoisomerase IIalpha, MIB-1, E2F, apoptotic index, APE/ref-1, p53, Her-2/neu, estrogen and porgesterone receptors, and histological grading) as predictors of pathologic response after anthracycline-based chemotherapy for breast cancer. A series of 50 consecutive breast cancer patients receiving anthracycline-based primary chemotherapy were retrospectively studied. Biological markers were assessed by immunohistochemistry (and by TUNEL assay for apoptotic index) in pre-treatment core biopsies and post-treatment surgical samples. The expression of topoisomerase IIalpha, E2F, MIB-1, estrogen and progesterone receptors decreased, while APE/ref-1 staining increased after treatment. Higher topoisomerase IIalpha (P=0.007) and lower APE/ref-1 (P=0.04) expression were associated with better pathologic response.

Expression of cell proliferation markers in pituitary adenomas--correlation and clinical relevance of MIB-1 and anti-topoisomerase-IIalpha.

Pituitary adenomas represent an inhomogeneous tumor entity in terms of growth rate, invasiveness and recurrence. To improve understanding of their different biological behaviour, tumor cell proliferation markers are applied. The aim of this study was to assess proliferation rates overall and in clinico-pathological subgroups using MIB-1 and the recently introduced cell proliferation marker anti-topoisomerase-IIalpha (Topo-IIalpha). Further, we correlated the two markers, and defined the clinical value of Topo-IIalpha in pituitary adenomas as compared to MIB-1. We analyzed tumor cell proliferation rates using MIB-1 and Topo-IIalpha antibodies on samples of 260 primary pituitary adenomas. We excluded recurrent cases and cases with drug pretreatment. Median patient age at the time of surgery was 47 years (range 14-86 years), the male:female ratio was 1:1. The total cohort comprised 110 non-functioning and 150 functioning cases. Subtyping was performed according to hormonal expression as defined by WHO. Tumor size and invasiveness were noted from surgical and/or radio logical reports in 95% of cases. Overall MIB-1 index was median 1.8% (range 0.2-23.6%), Topo-IIalpha index was median 1.0% (range 0-14.4%) with a strong correlation between the two markers ( R=0.837, P<0.001). As compared to MIB-1, mean Topo-IIalpha values were significantly lower by a factor 1.8. Only MIB-1 was significantly higher in invasive as compared to non-invasive adenomas, in tumors < or =3 cm in diameter, and in the age-group 21-40. Female gender had significantly higher MIB-1 and Topo-IIalpha indices than male. Silent ACTH-cell and PRL-producing adenomas had the highest, null-cell adenomas and gonadotropinomas the lowest proliferation values, respectively. Our data show a strong correlation between MIB-1 and Topo-IIalpha indices in pituitary adenomas. Only MIB-1 but not Topo-IIalpha demonstrated significantly higher values in invasive adenomas. Therefore, MIB-1 seems more useful than Topo-IIalpha for decisions regarding postoperative patient management.

Autoantibodies against IA-2, GAD, and topoisomerase II in type 1 diabetic patients.

Prevalence of autoantibodies against IA-2 (IA-2A), glutamic acid decarboxylase (GADA), and type II DNA topoisomerase (TopIIA) of Taiwanese type 1 diabetes mellitus (T1DM) patients was investigated. Correlations of these autoantibodies with patients' clinical manifestations were also analyzed. Prevalence of IA-2A, GADA, and TopIIA in our patients was 23.6%, 47.1%, and 55.2%, respectively. Eighty percent of the IA-2A recognized the carboxyl terminus of the IA-2 protein tyrosine phosphatase-like domain. Average disease duration of IA-2A+ patients was significantly shorter than that of IA-2A- patients [3.76+/-0.42 vs. 4.98+/-0.34 years, p = 0.028]. Presence of GADA was correlated with the mean age of onset [10.82+/-0.76 vs. 8.38+/-0.77 years for GADA+ and GADA- patients, p = 0.026]. Patients with adolescent onset have higher GADA prevalence and better residual beta-cell functions. TopIIA and GADA are suggested to be better markers for Taiwanese T1DM patients because of their higher prevalence and persistence.

Serum biomarkers of hepatitis B virus infected liver inflammation: a proteomic study.

Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin beta and alpha2 chain, apolipoprotein A-I and A-IV, alpha1-antitrypsin, transthyretin and DNA topoisomerase IIbeta. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and alpha1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.

Phase I evaluation of sequential topoisomerase targeting with irinotecan/cisplatin followed by etoposide in patients with advanced malignancy.

PURPOSE: To investigate pharmacologically guided addition of etoposide to a weekly irinotecan/cisplatin chemotherapy. PATIENTS AND METHODS: Patients with advanced nonhematologic malignancies were eligible. Treatment consisted of i.v. administration of 50 mg/m(2) irinotecan and 20 mg/m(2) cisplatin on days 1, 8, 15, and 22 of a 42-day cycle or on days 1 and 8 of a 21-day cycle. Etoposide was administered in a dose-escalating fashion 2 days after each dose of irinotecan/cisplatin, either i.v. as a single dose or p.o. as two doses administered 12 h apart. Pharmacologic analyses included measurement of plasma concentrations of irinotecan, SN-38, and SN-38 glucuronide, as well as quantitation of topoisomerase protein levels in peripheral blood mononuclear cells (PBMNCs). RESULTS: A total of 40 patients with a variety of malignancies received 122 cycles of therapy. Dose-limiting toxicities included neutropenia and diarrhea, with the 21-day cycle tolerated better than the 42-day cycle. For the 21-day cycle, the maximum tolerated dose was 75 mg/m(2) for i.v. etoposide and 85 mg/m(2) for oral etoposide. Objective responses were observed in four patients with previously treated mesothelioma, gastric, breast, and ovarian cancer, respectively. PBMNC levels of topoisomerase IIalpha were increased at the time of etoposide administration in two patients, with these patients having the highest SN-38 glucuronide peak-plasma-concentration and area-under-the-curve values among 15 patients with available pharmacokinetic data. One of these patients had a partial response to therapy. CONCLUSIONS: Pharmacologically guided administration of etoposide in combination with irinotecan/cisplatin using a 21-day cycle is associated with acceptable toxicity and significant antitumor activity. The finding that PBMNC topoisomerase IIalpha protein levels increased after irinotecan/cisplatin treatment in two of six patients supports the continued development of sequential topoisomerase targeting in the treatment of malignancy.

Drug resistance mechanisms in chronic lymphocytic leukemia.

Peripheral blood samples from 18 patients with chronic lymphocytic leukemias (CLL) who were either untreated but who were later sensitive to chlorambucil (CLL S) or resistant to a combination containing doxorubicin, vincristine, cyclophosphamide and prednisone (CLL R) were studied for glutathione system, P-glycoprotein, PCNA and topoisomerase II expression. P-glycoprotein expression detected by an immunocytochemical technique using MRK 16 antibody was present at the same level in CLL S and CLL R. The percentage of cells positive for P-gp was below 5% in all samples tested. Topoisomerase IIalpha level was quantified by Western blot analysis. None of the 18 CLL samples had detectable topoisomerase IIalpha protein. In addition, 12 CLL were tested for PCNA staining and no samples had more than 1% of positive cells at immunocytochemical detection indicating that CLL cells were not engaged in the cell cycle. Some differences were found between CLL S and CLL R in the glutathione system. Glutathione concentration (GSH) and GST activity was the same in CLL S and CLL R. The glutathione-S-transferase (GST) isoenzyme profile was different in the two CLL groups. The mean GST-pi and GST-alpha quantitation were twice as high as in CLL R compared to CLL S, but this difference did not reach statistical significance because of large variations between CLL samples. A significant correlation was observed between GST-pi expression and GST activity using CDNB as the substrate. GST-mu was detected in only one of seven CLL before therapy and in six of 11 resistant to chemotherapy. No correlation was found between P-glycoprotein expression, GST activity and the different GST isoenzymes studied. These results suggest that the glutathione system could play a role in the resistance of anticancer agents in chronic lymphocytic leukemia. The role of the other drug resistance mechanisms (P-glycoprotein and topoisomerase IIalpha) seems to be of limited importance.

A phase I and pharmacokinetic study of a new camptothecin derivative, 9-aminocamptothecin.

Camptothecins are the only available antitumor agents which target the nuclear enzyme topoisomerase I. 9-Aminocamptothecin (9-AC) is a water-insoluble derivative of camptothecin which has demonstrated impressive antitumor activity in preclinical models. While two other water-soluble derivatives, CPT-11 and topotecan, have successfully completed Phase I and Phase II testing, biochemical and tissue culture studies suggest that camptothecin analogues differ in characteristics which may be important in determining antitumor activity. We performed a Phase I trial of 9-AC to determine the pharmacokinetics, dose-limiting toxicity, and maximum tolerated dose of this agent when administered as a 72-h continuous i.v. infusion. Thirty-one patients with resistant solid cancers received 5-60 microgram/m2/h 9-AC for 72 h, repeated at 3-week intervals. The drug was administered in a vehicle containing dimethylacetamide, polyethylene glycol, and phosphoric acid. Blood samples were collected and the lactone (closed ring) form of 9-AC was quantitated. The maximum tolerated dose of 9-AC was determined to be 45 microgram/m2/h. Dose-limiting toxicity consisted of neutropenia. Thrombocytopenia was also prominent. There were no significant nonhematological toxicities. Minimal responses were seen in patients with gastric, colon, and non-small cell lung cancer. Although significant interpatient variation in plasma 9-AC lactone levels was observed, pooled data were fit to a two-compartment model, with a terminal half-life of 36 h. Analyses of topoisomerase protein levels in peripheral blood cells indicated decreases in topoisomerase I accompanied by increases in topoisomerase II in two of three patients. 9-AC is an active antitumor agent and may be administered safely as a 72-h infusion in patients with cancer. Although Phase II trials with a 72-h infusion of 9-AC are warranted, alternate schedules should be evaluated given the dramatic preclinical activity seen with more prolonged administrations.

Topoisomerase II expression in normal haemopoietic cells and chronic lymphocytic leukaemia: drug sensitivity or resistance?

Chronic lymphocytic leukaemia (CLL) is a progressive disease, commonly treated in its early stage with alkylating agents. A multi-agent regimen which includes anthracyclines is used to treat advanced disease. Despite chemotherapy, the disease remains incurable. There is now considerable evidence to suggest that anthracyclines exert their effect via the nuclear enzyme topoisomerase II and that alterations in amount or activity of this enzyme may mediate drug resistance. We have investigated topoisomerase II mRNA expression in 34 CLL patients and in haemopoietic cells from 10 normal donors. Expression was found to be low but detectable in all patients and normals. Such low levels may contribute to the toxicity of alkylating agents, but could severely limit the effect of anthracyclines.

DNase I hypersensitivity is independent of endogenous topoisomerase II activity during chicken erythrocyte differentiation.

Endogenous topoisomerase II cleavage sites were mapped in the chicken beta A-globin gene of 12- to 14-day embryonic erythrocytes. A major topoisomerase II catalytic site was mapped to the 5' end of the globin gene which contained a nucleosome-free and DNase I-hypersensitive site and additional but minor sites were mapped to the second intron and 3' of the gene to a tissue-specific enhancer. Cleavage sites, mapped in situ by indirect end labeling, were aligned to single-base-pair resolution by comparison to a consensus sequence derived for vertebrate topoisomerase II catalytic sites. In contrast to embryonic erythrocytes, endogenous topoisomerase II cleavages were not detected in erythrocytes from peripheral blood of adult chickens; therefore, as the transcriptional activity of the beta A-globin gene declines during terminal differentiation of erythrocytes, the activity of topoisomerase II in situ declines as well, despite the fact that DNase I hypersensitivity persists. The results showed that DNase I-hypersensitive chromatin can be maintained in the absence of topoisomerase II activity and suggested that topoisomerase II acts at hypersensitive sites because of an inherent attraction to some preexisting combination of DNA sequence or chromatin structure associated with DNase I-hypersensitive regions.