Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action.

We have examined whether inhibition of phosphatidylinositol-3 kinase (PI3K) and its target, the serine/threonine kinase Akt, play a role in the antitumor effect of the HER2 antibody Herceptin. Herceptin inhibited colony formation, down-regulated cyclin D1, and increased p27 protein levels in the HER2 gene-amplified BT-474 and SKBR-3 human breast cancer cells. These effects were temporally associated with the inhibition of PI3K activity in vitro as well as Akt function as measured by steady-state levels of phospho-Ser473 Akt and kinase activity against glycogen synthase kinase (GSK)-3beta. These responses were not observed in MDA-361 and MDA-453 cells, which do not exhibit HER2 gene amplification and are relatively resistant to Herceptin. Treatment of BT-474 cells with Herceptin inhibited the constitutive tyrosine phosphorylation of HER3 and disrupted the basal association of HER3 with HER2 and of HER3 with p85alpha potentially explaining the inhibition of PI3K. Treatment with either Herceptin or the PI3K inhibitor LY294002 increased the levels of p27 in the nucleus>cytosol, thus increasing the ratio of p27:Cdk2 in the nucleus and inhibiting Cdk2 activity and cell proliferation. Antisense p27 oligonucleotides abrogated the increase in p27 induced by Herceptin and prevented the antibody-mediated reduction in S phase. Transduction of BT-474 cells with an adenovirus-encoding active (myristoylated) Akt (Myr-Akt), but not with a beta-galactosidase control adenovirus, prevented the Herceptin- or LY294002-induced down-regulation of cyclin D1 and of phosphorylated GSK-3beta and prevented the accumulation of p27 in the nucleus and cytosol. In addition, Myr-Akt prevented Herceptin-induced inhibition of the cell proliferation of BT-474 cells and Herceptin-induced apoptosis of SKBR-3 cells. These data suggest that (a) changes in cell cycle- and apoptosis-regulatory molecules after HER2 blockade with Herceptin result, at least in part, from the inhibition of Akt; and (b) disabling PI3K and Akt is required for the antitumor effect of HER2 inhibitors.

Amplification and overexpression of topoisomerase IIalpha predict response to anthracycline-based therapy in locally advanced breast cancer.

PURPOSE: The putative association between erbB-2 overexpression and favorable response to anthracyline-based therapy in breast cancer is controversial, and the mechanism unclear. We sought to determine whether coamplification and overexpression of the topoisomerase IIalpha gene, near erbB-2 on chromosome 17, and a known anthracycline target, may underlie the association. EXPERIMENTAL DESIGN: Thirty-five patients who had locally advanced breast cancer (LABC) and who had received neoadjuvant, anthracycline-based therapy were studied. Copy number of topoisomerase IIalpha and erbB-2 was determined by fluorescence in situ hybridization, and expression by immunohistochemistry. RESULTS: Of 8 patients with erbB-2 amplification, 5 had a complete response (CR) or minimal residual disease (MRD), 3 had a partial response (PR), and none had stable (StD) or progressive disease (PD) at the time of mastectomy, versus 3 CR or MRD, 16 PR, and 8 StD or PD for patients without amplification (P = 0.008). In contrast, erbB-2 overexpression was not significantly associated with response (P = 0.114). Of 6 patients with topoisomerase IIalpha amplification, 4 had CR or MRD, 2 PR, and none StD or PD, versus 4 CR or MRD, 17 PR, and 8 StD or PD for patients without amplification (P = 0.034). All of the tumors with topoisomerase IIalpha amplification also had erbB-2 amplification, but not vice versa. Overexpression of topoisomerase IIalpha (9 patients) was also associated with favorable response (P = 0.021). CONCLUSIONS: Coamplification of erbB-2 and topoisomerase IIalpha is significantly associated with favorable local response to anthracycline-based therapy in LABC. The expression data favor a plausible mechanism based on topoisomerase IIalpha biology.

Detection of chromosomal instability in paired breast surgery and ductal lavage specimens by interphase fluorescence in situ hybridization.

PURPOSE: Ductal lavage is a new modality for collecting exfoliated breast cells with the goal of detecting early neoplasia. The purpose of our study was to evaluate the correlation between cancer-associated abnormalities in breast lesions and exfoliated breast cells collected by ductal lavage. EXPERIMENTAL DESIGN: We performed histopathologic, cytologic, and molecular cytogenetic analyses on 39 paired cases of surgically excised breast lesions and ductal lavage specimens collected immediately before surgery. RESULTS: Abnormal cytology was detected in 7 of 15 (47%) of the evaluable lavages collected from malignant cases, versus 4 of 19 (21%) of the evaluable lavages harvested from benign cases for a sensitivity and specificity of 47 and 79%, respectively. Interphase fluorescence in situ hybridization analysis of all evaluable lavages revealed numeric changes on chromosomes 1, 8, 11, and/or 17 in 10 of 14 (71%) specimens from malignant cases versus 2 of 18 (11%) from benign cases for a sensitivity and specificity of 71 and 89%, respectively. CONCLUSIONS: Our study demonstrates that cytologic and genetic abnormalities associated with breast cancer progression can be detected in ductal lavage cells collected from women with in situ and invasive breast cancer and suggests that fluorescence in situ hybridization may have superior sensitivity and specificity compared with conventional cytology.

Cyclin E overexpression and amplification in human tumours.

Cyclin E amplification and overexpression have recently been described in several tumour types. However, many tumour entities have never been examined for cyclin E alterations. Numerous and time-consuming experiments were previously required to determine the significance of potential oncogenes across different tumour types. To overcome this problem, tissue microarrays (TMAs) consisting of 3670 primary tumours from 128 different tumour types, 709 metastases, and 354 normal tissues were generated. Cyclin E alterations were then analysed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Cyclin E gene amplification was observed in 15 different tumour types and subtypes, ie rhabdomyosarcoma, urinary bladder cancer (three subtypes), ovarian cancer (two subtypes), malignant fibrous histiocytoma, adenocarcinoma of the small intestine, medullary breast cancer, gall bladder adenocarcinoma, phaeochromocytoma, gastric adenocarcinoma, squamous cell carcinoma of the uterine cervix, colonic adenocarcinoma, and endometrial carcinoma. Cyclin E protein accumulation was found in 48 different tumour types. The use of TMA technology has enabled us to expand considerably our knowledge of cyclin E alterations in human tumours. The occurrence of amplification and overexpression in many different tumour types suggests that cyclin E plays an important role in tumour biology.

A fluorescence in situ hybridization-based assay for improved detection of lung cancer cells in bronchial washing specimens.

BACKGROUND: Interphase fluorescence in situ hybridization (FISH) is a powerful tool for detecting chromosome and locus-specific changes in tumor cells. We developed a FISH-based assay to detect genetic changes in bronchial washing specimens of lung carcinoma patients. METHODS: The assay uses a mixture of fluorescently labeled probes to the centromeric region of chromosome 1 and to the 5p15, 8q24 (site of the c-myc gene), and 7p12 (site of the EGFR gene) loci to assess cells in bronchial washing specimens for chromosomal abnormalities indicative of lung carcinoma. The FISH assay was performed on 74 specimens that had been assessed previously for evidence of malignancy by routine cytology with Pap staining. RESULTS: Forty-eight patients had histologically confirmed lung carcinoma and 26 patients had a clinical diagnosis that was negative for lung carcinoma. FISH analysis was performed without knowledge of the patient's clinical information. The finding of six or more epithelial cells with gains of two or more chromosome regions was considered a positive FISH result (i.e., evidence of malignancy). The sensitivity of FISH for the detection of lung carcinoma was 82% in this set of specimens compared with a 54% sensitivity by design for cytology (FISH vs. cytology, P = 0.007). FISH detected 15 of 18 specimens that were falsely negative by cytology. The specificities of FISH and cytology were 82% and 100%, respectively, and were not significantly different (P = 0.993). CONCLUSIONS: The data indicate a potential utility of the FISH assay as an adjunct to bronchial washing cytology in routine clinical practice.

A comparison of BTA stat, hemoglobin dipstick, telomerase and Vysis UroVysion assays for the detection of urothelial carcinoma in urine.

PURPOSE: We determine the sensitivity and specificity of various assays for the detection of urothelial carcinoma. MATERIALS AND METHODS: A total of 280 voided urine specimens from 265 patients were obtained immediately before cystoscopy for BTA stat, (Bard Diagnostic, Redmond, Washington) hemoglobin dipstick, (Bayer, Elkhart, Indiana) telomerase and UroVysion (Vysis, a wholly owned subsidiary of Abbott Laboratories, Abbott Park, Illinois) analysis. RESULTS: Of the 265 patients 75 had biopsy proven urothelial carcinoma, and the sensitivity of the assays was determined from these patients. From most sensitive to least sensitive, the overall sensitivity of UroVysion (73 cases), BTA stat (72), hemoglobin dipstick (73) and telomerase (70) was 81%, 78%, 74%, and 46%, respectively. Each of the first 3 tests was statistically significantly more sensitive than the telomerase assay (p <0.05). However, the differences in overall sensitivity of UroVysion, BTA stat and hemoglobin dipstick were not statistically significant. The specificity of the tests was calculated for 80 of the 265 patients in this study who had no history of urothelial carcinoma and negative cystoscopy findings despite common urological complaints. From most specific to least specific, the specificity of UroVysion, telomerase, BTA stat and hemoglobin dipstick was 96%, 91%, 74% and 51%, respectively. UroVysion and telomerase were statistically significantly (p <0.01) more specific than the BTA stat and hemoglobin dipstick assays, and all of the assays were more specific than hemoglobin dipstick testing (p <0.001). CONCLUSIONS: Our study reveals that UroVysion is the most sensitive and specific assay among those tested for the detection of urothelial carcinoma. Telomerase testing had good specificity but poor sensitivity. The BTA stat and hemoglobin dipstick tests had good sensitivity but relatively poor specificity. UroVysion is a promising new assay for the detection of urothelial carcinoma in urine specimens. However, further studies are needed to explore the role of the various assays in the treatment of patients with superficial urothelial carcinoma.