Breast Cancer Index and prediction of benefit from extended endocrine therapy in breast cancer patients treated in the Adjuvant Tamoxifen-To Offer More? (aTTom) trial.

BACKGROUND: Extending the duration of adjuvant endocrine therapy reduces the risk of recurrence in a subset of women with early-stage hormone receptor-positive (HR+) breast cancer. Validated predictive biomarkers of endocrine response could significantly improve patient selection for extended therapy. Breast cancer index (BCI) [HOXB13/IL17BR ratio (H/I)] was evaluated for its ability to predict benefit from extended endocrine therapy in patients previously randomized in the Adjuvant Tamoxifen-To Offer More? (aTTom) trial. PATIENTS AND METHODS: Trans-aTTom is a multi-institutional, prospective-retrospective study in patients with available formalin-fixed paraffin-embedded primary tumor blocks. BCI testing and central determination of estrogen receptor (ER) and progesterone receptor (PR) status by immunohistochemistry were carried out blinded to clinical outcome. Survival endpoints were evaluated using Kaplan-Meier analysis and Cox regression with recurrence-free interval (RFI) as the primary endpoint. Interaction between extended endocrine therapy and BCI (H/I) was assessed using the likelihood ratio test. RESULTS: Of 583 HR+, N+ patients analyzed, 49% classified as BCI (H/I)-High derived a significant benefit from 10 versus 5 years of tamoxifen treatment [hazard ratio (HR): 0.35; 95% confidence interval (CI) 0.15-0.86; 10.2% absolute risk reduction based on RFI, P = 0.027]. BCI (H/I)-low patients showed no significant benefit from extended endocrine therapy (HR: 1.07; 95% CI 0.69-1.65; -0.2% absolute risk reduction; P = 0.768). Continuous BCI (H/I) levels predicted the magnitude of benefit from extended tamoxifen, whereas centralized ER and PR did not. Interaction between extended tamoxifen treatment and BCI (H/I) was statistically significant (P = 0.012), adjusting for clinicopathological factors. CONCLUSION: BCI by high H/I expression was predictive of endocrine response and identified a subset of HR+, N+ patients with significant benefit from 10 versus 5 years of tamoxifen therapy. These data provide further validation, consistent with previous MA.17 data, establishing level 1B evidence for BCI as a predictive biomarker of benefit from extended endocrine therapy. TRIAL REGISTRATION: ISRCTN17222211; NCT00003678.

Oestrogen receptor co-activator AIB1 is a marker of tamoxifen benefit in postmenopausal breast cancer.

BACKGROUND: The oestrogen receptor (ER) co-activator amplified in breast cancer 1 (AIB1) has been suggested as a treatment predictive and prognostic marker in breast cancer. Studies have however not been unanimous. PATIENTS AND METHODS: AIB1 protein expression was analysed by immunohistochemistry on tissue micro-arrays with tumour samples from 910 postmenopausal women randomised to tamoxifen treatment or no adjuvant treatment. Associations between AIB1 expression, clinical outcome in the two arms and other clinicopathological variables were examined. RESULTS: In patients with ER-positive breast cancer expressing low tumour levels of AIB1 (<75%), we found no significant difference in recurrence-free survival (RFS) or breast cancer-specific survival (BCS) between tamoxifen treated and untreated patients. In patients with high AIB1 expression (>75%), there was a significant decrease in recurrence rate (HR 0.40, 95% CI 0.26-0.61, P < 0.001) and breast cancer mortality rate (HR 0.38, 95% CI 0.21-0.69, P = 0.0015) with tamoxifen treatment. In the untreated arm, we found high expression of AIB1 to be significantly associated with lower RFS (HR 1.74, 95% CI 1.20-2.53, P = 0.0038). CONCLUSION: Our results suggest that high AIB1 is a predictive marker of good response to tamoxifen treatment in postmenopausal women and a prognostic marker of decreased RFS in systemically untreated patients.

Prognostic utility of HOXB13:IL17BR and molecular grade index in early-stage breast cancer patients from the Stockholm trial.

BACKGROUND: A dichotomous index combining two gene expression assays, HOXB13:IL17BR (H:I) and molecular grade index (MGI), was developed to assess risk of recurrence in breast cancer patients. The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer. METHODS: In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patients from the randomised prospective Stockholm trial, H:I and MGI were measured using real-time RT-PCR. Association with patient outcome was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression. A continuous risk index was developed using Cox modelling. RESULTS: The dichotomous H:I+MGI was significantly associated with distant recurrence and breast cancer death. The >50% of tamoxifen-treated patients categorised as low-risk had <3% 10-year distant recurrence risk. A continuous risk model (Breast Cancer Index (BCI)) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53% of patients categorised as low risk with an 8.3% 10-year distant recurrence risk. CONCLUSION: Retrospective analysis of this randomised, prospective trial cohort validated the prognostic utility of H:I+MGI and was used to develop and test a continuous risk model that enables prediction of distant recurrence risk at the patient level.

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.

In vivo gene expression profile analysis of human breast cancer progression.

The development and use of molecular-based therapy for breast cancer and other human malignancies will require a detailed molecular genetic analysis of patient tissues. The recent development of laser capture microdissection and high density cDNA arrays now provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in the actual neoplastic tissue milieu. We report the combined use of laser capture microdissection and high-throughput cDNA microarrays to monitor in vivo gene expression levels in purified normal, invasive, and metastatic breast cell populations from a single patient. These in vivo gene expression profiles were verified by real-time quantitative PCR and immunohistochemistry. The combined use of laser capture microdissection and cDNA microarray analysis provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of breast cancer and is generally applicable to the study of malignancy.

Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome.

Li Fraumeni Syndrome (LFS) is a multicancer phenotype, most commonly associated with germ-line mutations in TP53. In a kindred with LFS without an inherited TP53 mutation, we have previously reported a truncating mutation (1100delC) in CHK2, encoding a kinase that phosphorylates p53 on Ser(20). Here, we describe a CHK2 missense mutation (R145W) in another LFS family. This mutation destabilizes the encoded protein, reducing its half-life from >120 min to 30 min. This effect is abrogated by treatment of cells with a proteosome inhibitor, suggesting that CHK2(R145W) is targeted through this degradation pathway. Both 1100delC and R145W germ-line mutations in CHK2 are associated with loss of the wild-type allele in the corresponding tumor specimens, and neither tumor harbors a somatic TP53 mutation. Our observations support the functional significance of CHK2 mutations in rare cases of LFS and suggest that such mutations may substitute for inactivation of TP53.

Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis.

Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction-validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan-Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields biologically meaningful outcomes by taking advantage of the existing knowledge of the physical and functional interactions between the predicted target genes. The resulting interaction maps provide useful information on signaling pathways cooperatively involved in clinically important features of the malignant cells, such as drug resistance.

Changes in gene expression in beta cells after islet isolation and transplantation using laser-capture microdissection.

AIMS/HYPOTHESIS: The process of islet isolation can cause chemical and mechanical injury to beta cells. In addition, hyperglycaemia after islet transplantation can compromise beta cell function. The aim of this experiment was to evaluate changes in gene expression in endogenous islets using laser-capture microdissection (LCM). MATERIALS AND METHODS: Islets from B6AF1 mice were studied in situ in the pancreas as well as those freshly isolated or cultured for 24 h. Fresh islets were transplanted under the kidney capsule of syngeneic diabetic (streptozocin-induced) and non-diabetic mice. Frozen sections from all the samples were prepared for LCM to obtain beta cell-enriched tissue; RNA was extracted and amplified using T7 polymerase. RT-PCR was used to assess expression of selected genes critical for beta cell function (Ins, Ipf1 [previously known as Pdx1], Slc2a2 [previously known as GLUT2] and Ldha) and the stress response (Hmox1 [previously known as HO-1], Gpx1, Tnfaip3 [previously known as A20] and Fas). Immunostaining was also performed. RESULTS: In freshly isolated and cultured islets, insulin and Ipf1 mRNA levels were decreased by 40% (compared with islets in situ), while stress genes were upregulated. Comparison between in situ pancreatic islets and engrafted beta cells of cured mice showed declines in Ipf1 expression. CONCLUSIONS/INTERPRETATION: Our experiment, the first report to investigate changes in gene expression in endogenous islets using LCM, indicate that beta cells following islet isolation and residing in a foreign graft environment have decreased expression of genes involved in insulin production and increased expression of stress genes. Our data suggest that an islet graft, even in successful transplantation, may be different from endogenous islets in gene expression.

Sialylation of the sialic acid binding lectin sialoadhesin regulates its ability to mediate cell adhesion.

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3-linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.

Effects of H-7 are not exclusively mediated through protein kinase C or the cyclic nucleotide-dependent kinases.

Culturing murine T cell tumor lines in the presence of the protein kinase inhibitor H-7 for 4 days led to their dependence on H-7 for maximal constitutive proliferation. Withdrawal of H-7 from H-7-conditioned cells led to inhibition of proliferation and cell death. The mechanism underlying this H-7 dependence does not appear to be related to clonal selection or to effects on protein kinase C or the cyclic nucleotide-dependent kinases. This suggests that all the effects of the widely used H-7 may not be completely understood, and that H-7 may be useful in the dissection of the complex patterns of growth regulation in T cell malignancies.

Dynamic echo-planar imaging of the breast: experience in diagnosing breast carcinoma and correlation with tumor angiogenesis.

PURPOSE: To correlate quantitative echo-planar magnetic resonance (MR) imaging measures of gadopentetate dimeglumine tumor uptake with histologic diagnoses and microvessel density (MVD) and to compare dynamic echo-planar imaging of breast lesions with conventional dynamic MR imaging techniques. MATERIALS AND METHODS: The study group comprised 63 patients (aged 13-70 years) with 71 breast lesions who underwent conventional and echo-planar MR imaging. The T1 values, change in gadopentetate dimeglumine concentration, and extraction-flow products were calculated with the echo-planar imaging data and were correlated with histologic findings and MVD estimates. Extraction-flow product data normalized to pectoral muscle gadopentetate dimeglumine concentration in invasive cancers was also correlated with MVD. RESULTS: On average, cancer T1 values were shorter than benign values, but there was substantial overlap between the two groups. Cancers had higher extraction-flow products than benign lesions (P < .001). Sensitivity, specificity, positive predictive value, and negative predictive value were 83%, 79%, 67%, and 90%, respectively. Receiver operating characteristic analysis showed improved performance with extraction-flow products than with percentages of signal intensity change. Among the invasive cancers, there was no significant correlation between extraction-flow product and MVD. CONCLUSION: The T1 value remains important in more precise quantitative estimation of gadopentetate dimeglumine uptake in breast tumors, which helps improve the specificity of dynamic imaging. Tumor MVD affects the contrast medium enhancement of breast lesions, but other factors contribute.

New approaches to proteomic analysis of breast cancer.

Proteomic based approaches are beginning to be utilized to study the natural history and treatment of breast cancer. A variety of proteomics approaches are under study, and are summarized herein. Two-dimensional gel electrophoresis (2D-PAGE) is still the foundation of most proteomics studies. We present an analysis of 2D-PAGE studies reported to date in breast cancer, including those examining normal/tumor differences and selected populations of breast cells. Newer technologies such as laser capture microdissection and highly sensitive mass spectrometry methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Novel technologies still in developmental phases will enable identification of validated targets in small biopsy specimens, including high density protein arrays, antibody arrays and lysate arrays. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells and may be best suited for clinical biomarker studies. We present SELDI-TOF data herein to show the accuracy of the method in a small cohort of breast tumors, as well as its potential discriminatory capability. Such technologies are expected to supplement our armamentarium of mRNA-based assays, and provide critical information on protein levels and post-translational modifications.

Benign and malignant breast lesions: differentiation with echo-planar MR imaging.

PURPOSE: To quantify dynamic enhancement of breast lesions with echo-planar and conventional magnetic resonance (MR) imaging, to correlate these data with histologic findings and vessel density, and to evaluate MATERIALS AND METHODS: Twenty female patients with 22 breast lesions underwent conventional and MR echo-planar imaging T1 values, change in gadopentetate dimeglumine concentration, and extraction-flow products were calculated with echo-planar imaging data and were correlated with histologic findings and microvessel density. RESULTS: T1 values of cancers were not statistically significantly shorter. Cancers had more rapid uptake and higher extraction-flow products (P < .02). Sensitivity was 86% and specificity was 93% for diagnosis of malignancy. Microvessel density was higher for malignant lesions (P < .02) with an overall positive (not statistically significant) correlation between extraction-flow product and microvessel density. CONCLUSION: Echo-planar imaging appears promising for quantification of breast lesion enhancement. Microvessel data indicate that tumor angiogenesis affects enhancement.

BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function.

BRCA1 interacts in vivo with a novel protein, BACH1, a member of the DEAH helicase family. BACH1 binds directly to the BRCT repeats of BRCA1. A BACH1 derivative, bearing a mutation in a residue that was essential for catalytic function in other helicases, interfered with normal double-strand break repair in a manner that was dependent on its BRCA1 binding function. Thus, BACH1/BRCA1 complex formation contributes to a key BRCA1 activity. In addition, germline BACH1 mutations affecting the helicase domain were detected in two early-onset breast cancer patients and not in 200 matched controls. Thus, it is conceivable that, like BRCA1, BACH1 is a target of germline cancer-inducing mutations.

Germ-line BRCA1 mutations in Jewish and non-Jewish women with early-onset breast cancer.

BACKGROUND: Mutations in a germ-line allele of the BRCA1 gene contribute to the familial breast cancer syndrome. However, the prevalence of these mutations is unknown in women with breast cancer who do not have the features of this familial syndrome. We sought BRCA1 mutations in women who were given a diagnosis of breast cancer at an early age, because early onset is characteristic of a genetic predisposition to cancer. METHODS: Clinical information and peripheral-blood mononuclear cells were obtained from 418 women from the Boston metropolitan area in whom breast cancer was diagnosed at or before the age of 40. A comprehensive BRCA1 mutational analysis, involving automated nucleotide sequencing and a protein-truncation assay, was undertaken in 30 of these women, who had breast cancer before the age of 30. In addition, the BRCA1 mutation 185delAG, which is prevalent in the Ashkenazi Jewish population, was sought with an allele-specific polymerase-chain-reaction assay in 39 Jewish women among the 418 women who had breast cancer at or before the age of 40. RESULTS: Among 30 women with breast cancer before the age of 30, 4 (13 percent) had definite, chain-terminating mutations and 1 had a missense mutation. Two of the four Jewish women in this cohort had the 185delAG mutation. Among the 39 Jewish women with breast cancer at or before the age of 40, 8 (21 percent) carried the 185delAG mutation (95 percent confidence interval, 9 to 36 percent). CONCLUSIONS: Germ-line BRCA1 mutations can be present in young women with breast cancer who do not belong to families with multiple affected members. The specific BRCA1 mutation known as 185delAG is strongly associated with the onset of breast cancer in Jewish women before the age of 40.